7-deazapurine modulators of histone methyltransferase, and methods of use thereof

ABSTRACT

Disclosed are compounds, pharmaceutical compositions containing the compounds, uses of the compounds and compositions as modulators of histone methyltransferases, and methods for treating diseases influenced by modulation of histone methyltransferase activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/991,328, which is a national stage application, filed under 35 U.S.C.§371, of International Application No. PCT/US2011/063314, filed Dec. 5,2011, which claims priority to, and the benefit of, U.S. provisionalapplication No. 61/419,504, filed Dec. 3, 2010, the contents of each ofwhich are incorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

In eukaryotic cells DNA is packaged with histones to form chromatin.Approximately 150 base pairs of DNA are wrapped twice around an octamerof histones (two each of histones 2A, 2B, 3 and 4) to form a nucleosome,the basic unit of chromatin. Changes in the ordered structure ofchromatin can lead to alterations in transcription of associated genes.This process is highly controlled because changes in gene expressionpatterns can profoundly affect fundamental cellular processes such asdifferentiation, proliferation and apoptosis. Control of changes inchromatin structure (and hence of transcription) is mediated by covalentmodifications to histones, most notably of their N-terminal tails. Thesemodifications are often referred to as epigenetic because they can leadto heritable changes in gene expression, but do not affect the sequenceof the DNA itself. Covalent modifications (for example, methylation,acetylation, phosphorylation and ubiquitination) of the side chains ofamino acids are enzymatically mediated.

The selective addition of methyl groups to specific amino acid sites onhistones is controlled by the action of a unique family of enzymes knownas histone methyltransferases (HMTs). The level of expression of aparticular gene is influenced by the presence or absence of a methylgroup at a relevant histone site. The specific effect of a methyl groupat a particular histone site persists until the methyl group is removedby a histone demethylase, or until the modified histone is replacedthrough nucleosome turnover. In a like manner, other enzyme classes candecorate DNA and histones with other chemical species, and still otherenzymes can remove these species to provide temporal control of geneexpression.

The orchestrated collection of biochemical systems behindtranscriptional regulation must be tightly controlled in order for cellgrowth and differentiation to proceed optimally. Disease states resultwhen these controls are disrupted by aberrant expression and/or activityof the enzymes responsible for DNA and histone modification. In humancancers, for example, there is a growing body of evidence to suggestthat dysregulated epigenetic enzyme activity contributes to theuncontrolled cell proliferation associated with cancer as well as othercancer-relevant phenotypes such as enhanced cell migration and invasion

Rearrangements of the mixed lineage leukemia (MLL) gene on chromosome11q23 are associated with aggressive leukemias with a poor prognosis.MLL translocations result in aberrant recruitment of DOT1L, a histonemethyltransferase that methylates lysine 79 of histone H3 (H3K79), tochromatin leading to ectopic H3K79 methylation and increased expressionof genes involved in leukemogenesis. These rearrangements, which arefound in over 70% of infant leukemias and approximately 10% of adultacute myeloid leukemias (AML), result in the expression of fusionproteins in which the C-terminal sequences of MLL, including aSET-domain that methylates lysine 4 of histone H3 (H3K4), are replacedwith sequences derived from a variety of fusion partners, including AF4,AF9, and ENL. The majority of these fusion partners are components oftranscriptional elongation complexes that, directly or indirectly,recruit DOT1L to genomic loci bound by the MLL-fusion protein. Thisresults in elevated H3K79 methylation and increased mRNA expression ofMLL-fusion target genes, such as HOXA9 and MEIS1 that are central to thepathogenesis of leukemia.

Mistargeted DOT1L enzymatic activity has therefore been proposed as adriver of disease in MLL patients, however in the absence of specificDOT1L methyltransferase inhibitors, this hypothesis has not beendirectly addressed in model systems.

Beyond cancer, there is growing evidence for a role of epigeneticenzymes in a number of other human diseases, including metabolicdiseases (such as diabetes), inflammatory diseases (such as Crohn'sdisease), neurodegenerative diseases (such as Alzheimer's disease) andcardiovascular diseases. Therefore, selectively modulating the aberrantaction of epigenetic enzymes holds great promise for the treatment of arange of diseases.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to compounds thatselectively modulate the activity of the histone methyltransferaseDOT1L. For example, one aspect of the invention relates to a compound offormula I:

or a pharmaceutically acceptable salt, hydrate, enantiomer orstereoisomer thereof, wherein independently for each occurrence,

R¹ is hydrogen, alkyl, cycloalkyl, alkylcycloalkyl, alkylaryl,haloalkyl, formyl, heterocyclyl, heterocyclylalkyl,

or (C₂-C₄)alkyl substituted with

except that when X is

R¹ is not

R¹⁰ is hydrogen or alkyl;

R^(11a) is hydrogen, alkyl, or alkyl-cycloalkyl;

R^(11b) is hydrogen or alkyl; or taken together with R^(11a) and thenitrogen to which it is attached forms a 4- to 8-membered heterocyclylcomprising 0 or 1 additional heteroatoms;

R¹³ is hydrogen, alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl orsilyl;

R¹⁴ is hydrogen, alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R¹⁵ is alkyl, cycloalkyl or cycloalkylalkyl;

R²⁰ is hydrogen, alkyl, cycloalkyl or cycloalkylalkyl;

R² is

Y is —NH—, —N(alkyl)-, —O—, or —CR⁶ ₂—;

R^(22a) is aryl, heteroaryl, aralkyl, heteroaralkyl, fused bicyclyl,biaryl, aryloxyaryl, heteroaryloxyaryl, aryloxyheteroaryl orheteroaryloxyheteroaryl;

R^(22b) is hydrogen or alkyl;

R²⁴ is hydrogen or alkyl;

R^(25a), R^(25b), R^(25c), and R^(25d) independently are -M₂-T₂, inwhich M₂ is a bond, SO₂, SO, S, CO, CO₂, O, O—C₁-C₄ alkyl linker, C₁-C₄alkyl linker, NH, or N(R_(t)), R_(t) being C₁-C₆ alkyl, and T₂ is H,halo, or R_(S4), R_(S4) being C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 8-membered heterocycloalkyl, or 5 to10-membered heteroaryl, and each of O—C₁-C₄ alkyl linker, C₁-C₄ alkyllinker, R_(t), and R_(S4) being optionally substituted with one or moresubstituents selected from the group consisting of halo, hydroxyl,carboxyl, cyano, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 6-membered heterocycloalkyl, and 5 to6-membered heteroaryl;

R³ is hydrogen, halogen, hydroxy, alkyloxy, aralkyloxy, alkylcarbonyloxyor silyloxy;

R⁴ is hydrogen, halogen, hydroxy, alkyloxy, aralkyloxy, alkylcarbonyloxyor silyloxy;

R⁴¹ is hydrogen, alkyl or alkynyl;

Z is hydrogen or

R^(5a) is hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aryl, heteroaryl, biaryl, alkenylalkyl, alkynylalkyl,carbocyclylalkyl, heterocyclylalkyl, aralkyl, heteroaralkyl,alkylcarbonylaminoalkyl, arylcarbonylaminoalkyl,aralkylcarbonylaminoalkyl, arylsulfonylaminoalkyl, alkylthioalkyl,aralkylthioalkyl or heteroaralkylthioalkyl; or alkyl substituted with 1,2 or 3 substituents independently selected from the group consisting ofhydroxy, halo, carboxy, alkyoxy, aryloxy, aralkyloxy, nitro, amino,amido, aryl and heteroaryl:

R^(5b) is hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aryl, heteroaryl, biaryl, alkenylalkyl, alkynylalkyl,carbocyclylalkyl, heterocyclylalkyl, aralkyl, heteroaralkyl,alkylcarbonylaminoalkyl, arylcarbonylaminoalkyl,aralkylcarbonylaminoalkyl, arylsulfonylaminoalkyl, alkylthioalkyl,aralkylthioalkyl or heteroaralkylthioalkyl; or alkyl substituted with 1,2 or 3 substituents independently selected from the group consisting ofhydroxy, halo, carboxy, alkyoxy, aryloxy, aralkyloxy, nitro, amino,amido, aryl and heteroaryl; or taken together with R^(5a) and thenitrogen to which it is attached forms a 4- to 8-membered heterocyclylcomprising 0 or 1 additional heteroatoms;

R⁶ is hydrogen, alkyl or halo; or two geminal R⁶ taken together areethylene, propylene or butylene;

R^(7a) is hydrogen, lower alkyl, lower haloalkyl, cyano, halo, loweralkoxy, or C₃-C₅ cycloalkyl, optionally substituted with 1, 2 or 3substituents independently selected from the group consisting of cyano,lower alkoxy and halo;

R^(7b) is hydrogen, lower alkyl, lower haloalkyl, cyano, halo, loweralkoxy, or C₃-C₅ cycloalkyl, optionally substituted with 1, 2 or 3substituents independently selected from the group consisting of cyano,lower alkoxy and halo; and

R^(7c) is hydrogen, lower alkyl, lower haloalkyl, cyano, halo, loweralkoxy, or C₃-C₅ cycloalkyl, optionally substituted with 1, 2 or 3substituents independently selected from the group consisting of cyano,lower alkoxy and halo.

Another aspect of the invention relates to a pharmaceutical compositioncomprising an compound of the invention (e.g., a compound of formula I,or a pharmaceutically acceptable salt, hydrate, enantiomer orstereoisomer thereof), and one or more pharmaceutically acceptablecarriers. A pharmaceutical composition of the invention may alsocomprise a second therapeutic agent. Such pharmaceutical compositions ofthe invention can be administered in accordance with a method of theinvention (for example, as part of a therapeutic regimen for treatmentor prevention of conditions and disorders related to cancer and/orneurodegenerative disorders). In one embodiment, the invention relatesto a packaged pharmaceutical comprising a therapeutically effectiveamount of the compound or composition. In one embodiment, the inventionrelates to a packaged pharmaceutical comprising a prophylacticallyeffective amount of the compound or composition.

Another aspect of the invention relates to a method of treating orpreventing a disorder in which DOT1-mediated protein methylation plays apart, comprising administering to a subject in need thereof atherapeutically effective amount of a compound of the present invention.Such methods can be used to ameliorate any condition which is caused byor potentiated by the activity of DOT1.

Another aspect of the invention relates to a method of inhibiting orreducing the level of DOT1L activity in a cell comprising the step ofcontacting a cell with or providing to a subject a compound of thepresent invention.

Another aspect of the invention relates to a method of inhibiting orreducing the level of histone H3 lysine residue 79 (H3K79) methylationin a cell comprising the step of contacting a cell with or providing toa subject a compound of the present invention. Such methods can be usedto ameliorate any condition which is caused by or potentiated by theactivity of DOT1 through H3K79 methylation.

Another aspect of the invention relates to a method of treating orpreventing specific disorders in which DOT1 methylation plays a part,for example, in cancer or a neurological disorder. Such methods comprisethe step of administering to a subject in need thereof a therapeuticallyeffective amount of a compound or pharmaceutical composition of theinvention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts general routes for preparing compounds of the invention.

FIG. 2 depicts general routes for preparing compounds of the invention.

FIG. 3 depicts a route to compound 8 and its hydrochloride salt 9.

FIG. 4 depicts a route to compound 16 and its hydrochloride salt 17.

FIG. 5 tabulates inhibition (IC₅₀ values) of human histonemethyltransferases (HMTs) by compound 16.

FIG. 6 tabulates concentration-dependent inhibition of cellproliferation by compound 16 in various cell types.

FIG. 7 depicts a route to compound 20.

FIG. 8 depicts a route to compound 21.

DETAILED DESCRIPTION

Underlying Molecular Biology

Chromatin structure is important in gene regulation and epigeneticinheritance. Post-translational modifications of histones are involvedin the establishment and maintenance of higher-order chromatinstructure; for example, the tails of certain core histones are modifiedby acetylation, methylation, phosphorylation, ribosylation andubiquitination.

One aspect of the present invention relates to compounds thatselectively modulate the activity of the histone methyltransferaseDOT1L, an enzyme known to methylate lysine 79 of histone H3 (“H3K79”) invivo (Feng et al. (2002) Curr. Biol. 12:1052-1058). Similar to otherHMTases, DOT1L contains a S-adenosylmethionine (SAM) binding site anduses SAM as a methyl donor. However, unlike other reported HMTases, theDOT1 polypeptides do not contain a SET domain.

DOT1L nucleic acid and polypeptides have previously been described (see,e.g., U.S. Patent Application Publication No. 2005-0048634 A1(incorporated by reference); Feng et al. (2002) Curr. Biol.12:1052-1058; and Okada et al. (2005) Cell 121:167-78). The yeasthomolog of DOT1 was originally identified as a Disruptor of Telomericsilencing (the protein and nucleic acid sequences of yeast DOT1 can befound at GenBank Accession No. NP010728, incorporated herein byreference in its entirety). The human DOT1 homolog has been cloned,isolated, and designated as hDOT1L (human DOT1-like protein). Thesequences of the human nucleic acid and protein have been depositedunder GenBank Accession No. AF509504, which is hereby incorporated byreference in its entirety. Only the approximately 360 N-terminal aminoacids of hDOT1L share significant sequence similarity with the yeastDOT1. In addition, DOT1 homologs from C. elegans (GenBank Accession Nos.NP510056 and CAA90610), Drosophila (GenBank Accession Nos. CG10272 andAAF54122), mouse (GenBank Accession No. XP125730), Anopheles gambiae(GenBank Accession No. EAA03558), and Neurospora crassa (GenBankAccession No. EAA33634) are available in public databases (thedisclosures of which are incorporated by reference herein in theirentireties). The SAM binding domain among these homologs is conserved(approximately 30-100% amino acid sequence identity and 50-100% aminoacid similarity). Various aspects of the present invention can bepracticed with any DOT1L polypeptide or nucleic acid.

The 2.5 angstrom resolution structure of a fragment of the hDOT1Lprotein containing the catalytic domain (amino acids 1-416) has beensolved; and the atomic coordinates for amino acids 1-416 of hDOT1L havebeen determined and deposited in the RCSB database under ID code 1NW3and described in the scientific literature (see Min, et al. (2003) Cell112:711-723), the disclosures of both of which are incorporated hereinby reference in their entireties.

It has recently been demonstrated that hDOT1L plays an important role inMLL-AF10-mediated leukemogenesis (Okada et al. (2005) Cell 121:167-78).It was also shown that mistargeting of hDOT1L to the Hoxa9 gene byMLL-AF10 results in H3K79 methylation and Hoxa9 upregulation whichcontributes to leukemic transformation (Okada et al. (2005) Cell121:167-78). It was further demonstrated that the hDOT1L and MLL-AF10interaction involves the OM-LZ (octapeptide motif-leucine zipper) regionof AF10, required for MLL-AF10-mediated leukemic transformation(DiMartino et al. (2002) Blood 99:3780-5). It has also been shown thatCALM-AF10 fusion appears to be both necessary and sufficient to mediateleukemogenesis in vitro and in vivo; that hDOT1L and its H3K79methyltransferase activity are implicated in CALM-AF10-mediated leukemictransformation; and that the Hoxa5 gene is involved inCALM-AF10-mediated transformation (U.S. Patent Application PublicationNo. 2009-0061443 A1, which is hereby incorporated by reference in itsentirety). Aberrant recruitment of DOT1L leading to deregulated geneexpression may be a common feature of many other oncogenic MLL-fusionproteins. For example, the MLL fusion partners ENL, AF4, and AF9 arenormally found in nuclear complexes with DOT1L (Bitoun et al. (2007)Hum. Mol. Genet. 16:92-106, Mueller et al. (2007) Blood 110:4445-54,Zhang et al. (2006) J. Biol. Chem. 281:18059-68), and altered H3K79methylation profiles are a feature of murine and human MLL-AF4 leukemias(Krivstov et al. (2008) Cancer Cell 14:355-368).

Compounds

One aspect of the invention relates to a compound of formula I:

or a pharmaceutically acceptable salt, hydrate, enantiomer orstereoisomer thereof, wherein independently for each occurrence,

R¹ is hydrogen, alkyl, cycloalkyl, alkylcycloalkyl, alkylaryl,haloalkyl, formyl, heterocyclyl, heterocyclylalkyl,

or (C₂-C₄) alkyl substituted with

except that when X is

R¹ is not

R¹⁰ is hydrogen or alkyl;

R^(11a) is hydrogen, alkyl, or alkyl-cycloalkyl;

R^(11b) is hydrogen or alkyl; or taken together with R^(11a) and thenitrogen to which it is attached forms a 4- to 8-membered heterocyclylcomprising 0 or 1 additional heteroatoms;

R¹³ is hydrogen, alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl orsilyl;

R¹⁴ is hydrogen, alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R¹⁵ is alkyl, cycloalkyl or cycloalkylalkyl;

R²⁰ is hydrogen, alkyl, cycloalkyl or cycloalkylalkyl;

R² is

Y is —NH—, —N(alkyl)-, —O—, or —CR⁶ ₂—;

R^(22a) is aryl, heteroaryl, aralkyl, heteroaralkyl, fused bicyclyl,biaryl, aryloxyaryl, heteroaryloxyaryl, aryloxyheteroaryl orheteroaryloxyheteroaryl;

R^(22b) is hydrogen or alkyl;

R²⁴ is hydrogen or alkyl;

R^(25a), R^(25b), R^(25c), and R^(25d) independently are -M₂-T₂, inwhich M₂ is a bond, SO₂, SO, S, CO, CO₂, O, O—C₁-C₄ alkyl linker, C₁-C₄alkyl linker, NH, or N(R_(t)), R_(t) being C₁-C₆ alkyl, and T₂ is H,halo, or R_(S4), R_(S4) being C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 8-membered heterocycloalkyl, or 5 to10-membered heteroaryl, and each of O—C₁-C₄ alkyl linker, C₁-C₄ alkyllinker, R_(t), and R_(S4) being optionally substituted with one or moresubstituents selected from the group consisting of halo, hydroxyl,carboxyl, cyano, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 6-membered heterocycloalkyl, and 5 to6-membered heteroaryl;

R³ is hydrogen, halogen, hydroxy, alkyloxy, aralkyloxy, alkylcarbonyloxyor silyloxy;

R⁴ is hydrogen, halogen, hydroxy, alkyloxy, aralkyloxy, alkylcarbonyloxyor silyloxy;

R⁴¹ is hydrogen, alkyl or alkynyl;

Z is hydrogen or

R^(5a) is hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aryl, heteroaryl, biaryl, alkenylalkyl, alkynylalkyl,carbocyclylalkyl, heterocyclylalkyl, aralkyl, heteroaralkyl,alkylcarbonylaminoalkyl, arylcarbonylaminoalkyl,aralkylcarbonylaminoalkyl, arylsulfonylaminoalkyl, alkylthioalkyl,aralkylthioalkyl or heteroaralkylthioalkyl; or alkyl substituted with 1,2 or 3 substituents independently selected from the group consisting ofhydroxy, halo, carboxy, alkyoxy, aryloxy, aralkyloxy, nitro, amino,amido, aryl and heteroaryl:

R^(5b) is hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aryl, heteroaryl, biaryl, alkenylalkyl, alkynylalkyl,carbocyclylalkyl, heterocyclylalkyl, aralkyl, heteroaralkyl,alkylcarbonylaminoalkyl, arylcarbonylaminoalkyl,aralkylcarbonylaminoalkyl, arylsulfonylaminoalkyl, alkylthioalkyl,aralkylthioalkyl or heteroaralkylthioalkyl; or alkyl substituted with 1,2 or 3 substituents independently selected from the group consisting ofhydroxy, halo, carboxy, alkyoxy, aryloxy, aralkyloxy, nitro, amino,amido, aryl and heteroaryl; or taken together with R^(5a) and thenitrogen to which it is attached forms a 4- to 8-membered heterocyclylcomprising 0 or 1 additional heteroatoms;

R⁶ is hydrogen, alkyl or halo; or two geminal R⁶ taken together areethylene, propylene or butylene;

R^(7a) is hydrogen, lower alkyl, lower haloalkyl, cyano, halo, loweralkoxy, or C₃-C₅ cycloalkyl, optionally substituted with 1, 2 or 3substituents independently selected from the group consisting of cyano,lower alkoxy and halo;

R^(7b) is hydrogen, lower alkyl, lower haloalkyl, cyano, halo, loweralkoxy, or C₃-C₅ cycloalkyl, optionally substituted with 1, 2 or 3substituents independently selected from the group consisting of cyano,lower alkoxy and halo; and

R^(7c) is hydrogen, lower alkyl, lower haloalkyl, cyano, halo, loweralkoxy, or C₃-C₅ cycloalkyl, optionally substituted with 1, 2 or 3substituents independently selected from the group consisting of cyano,lower alkoxy and halo.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein X is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein X is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein X is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein X is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein X is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein X is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein X is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R² is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R² is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R²⁴ is hydrogen or alkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R²⁴ is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R^(25a) is hydrogen, alkyl, —O-alkyl,halogen, trifluoroalkyl, —O-trifluoromethyl, or —SO₂-trifluoromethyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R^(25b) hydrogen, alkyl, halogen, ortrifluoroalkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R^(25c) is hydrogen, alkyl, orhalogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R^(25c) is hydrogen or halogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R² is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein Y is —NH— or —N(alkyl)-.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein Y is —NH—.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein Y is —N(CH₃)—.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein Y is —O—.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein Y is —CH₂—.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R^(22a) is aryl or aralkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R^(22a) is substituted phenyl orsubstituted benzyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R^(22a) is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R^(22a) is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R^(22b) is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R^(22b) is methyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is alkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is —CH₃, —CH₂CH³, —CH₂CH(CH₃)₂ or—CH₂CH₂CH(CH₃)₂.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is C₃-C₇ cycloalkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is cyclopropyl, cyclopropylmethyl,2-cyclopropylethyl, cyclobutyl, cyclobutylmethyl, 2-cyclobutylethyl,cyclopentyl, cyclopentylmethyl, or 2-cyclopentylethyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is —CH₂CF₃.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is —CH₂Ph.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is —C(═O)H.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is —C(═O)CH₃.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is heterocyclyl orheterocyclylalkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹⁵ is alkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹⁵ is cycloalkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹⁵ is cycloalkylalkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is (C₂-C₄) alkyl substituted with

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R^(11a) is hydrogen, alkyl, oralkyl-cycloalkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R^(11a) is hydrogen, methyl, ori-propyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹³ is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein A is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein A is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein A is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein A is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein A is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein A is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein A is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein A is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein A is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein A is

and R⁶ is alkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein A is

and R⁶ is methyl, ethyl or isopropyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R³ is hydroxyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R³ is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R⁴ is hydroxyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R⁴ is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R⁴¹ is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R⁴¹ is methyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R³ is hydroxyl; and R⁴ is hydroxyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R³ is hydroxyl; R⁴ is hydroxyl; andR⁴¹ is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R³ is hydroxyl; R⁴ is hydroxyl; andR⁴¹ is methyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R³ is hydrogen; and R⁴ is hydroxyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R³ is hydrogen; R⁴ is hydroxyl; andR⁴¹ is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R³ is hydrogen; R⁴ is hydroxyl; andR⁴¹ is methyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R³ is hydroxyl; and R⁴ is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R³ is hydroxyl; R⁴ is hydrogen; andR⁴¹ is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R³ is hydroxyl; R⁴ is hydrogen; andR⁴¹ is methyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein Z is hydrogen or

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein Z is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein Z is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R^(5a) is hydrogen, alkyl,carbocyclyl, heterocyclyl, aryl, heteroaryl, carbocyclylalkyl,heterocyclylalkyl, aralkyl, or heteroaralkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R^(5a) is hydrogen, aralkyloxyalkyl,alkyl, aryl, aralkyl, aminoalkyl or hydroalkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R^(5a) is —H, —CH₂CH₂OCH₂Ph, —CH₂CH₃,—CH(CH₃)₂, -Ph, —CH₂CH(CH₃), —CH₂Ph, —CH₂CH₂NH₂, —CH₂(cyclohexyl) or—CH₂CH₂OH.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R^(5b) is hydrogen, alkyl,carbocyclyl, heterocyclyl, aryl, heteroaryl, carbocyclylalkyl,heterocyclylalkyl, aralkyl, or heteroaralkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R^(5b) is hydrogen, aralkyloxyalkyl,alkyl, aryl, aralkyl, aminoalkyl or hydroalkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R^(5b) is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R^(5a) is —H, —CH₂CH₂OCH₂Ph, —CH₂CH₃,—CH(CH₃)₂, -Ph, —CH₂CH(CH₃), —CH₃, —CH₂Ph, —CH₂CH₂NH₂, —CH₂(cyclohexyl)or —CH₂CH₂OH; and R^(5b) is —H.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R⁷ is hydrogen or lower alkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R^(7a) is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R^(7b) is hydrogen or lower alkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R^(7b) is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R^(7c) is hydrogen or lower alkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R^(7c) is hydrogen.

One aspect of the invention relates to a compound, or a pharmaceuticallyacceptable salt, hydrate, enantiomer or stereoisomer thereof, isselected from the group consisting of

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein the compound inhibits DOT1L with anIC₅₀ of less than about 10 μM. In certain embodiments, the inventionrelates to any one of the aforementioned compounds, wherein the compoundinhibits DOT1L with an IC₅₀ of less than about 5 μM. In certainembodiments, the invention relates to any one of the aforementionedcompounds, wherein the compound inhibits DOT1L with an IC₅₀ of less thanabout 1 μM. In certain embodiments, the invention relates to any one ofthe aforementioned compounds, wherein the compound inhibits DOT1L withan IC₅₀ of less than about 750 nM. In certain embodiments, the inventionrelates to any one of the aforementioned compounds, wherein the compoundinhibits DOT1L with an IC₅₀ of less than about 500 nM. In certainembodiments, the invention relates to any one of the aforementionedcompounds, wherein the compound inhibits DOT1L with an IC₅₀ of less thanabout 250 nM. In certain embodiments, the invention relates to any oneof the aforementioned compounds, wherein the compound inhibits DOT1Lwith an IC₅₀ of less than about 100 nM.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein the compound is a selective inhibitorof DOT1L.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein the compound inhibits both DOT1L andEZH2; the compound has a DOT1L IC₅₀ of between about 0.001 μM and about10 μM; and the ratio of the EZH2 IC₅₀ to the DOT1L IC₅₀ is between about10 and about 50. In certain embodiments, the invention relates to anyone of the aforementioned compounds, wherein the compound inhibits bothDOT1L and EZH2; the compound has a DOT1L IC₅₀ of between about 0.001 μMand about 10 μM; and the ratio of the EZH2 IC₅₀ to the DOT1L IC₅₀ isbetween about 50 and about 100. In certain embodiments, the inventionrelates to any one of the aforementioned compounds, wherein the compoundinhibits both DOT1L and EZH2; the compound has a DOT1L IC₅₀ of betweenabout 0.001 μM and about 10 μM; and the ratio of the EZH2 IC₅₀ to theDOT1L IC₅₀ is between about 100 and about 1,000.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein the compound inhibits both DOT1L andEHMT2; the compound has a DOT1L IC₅₀ of between about 0.001 μM and about10 μM; and the ratio of the EHMT2 IC₅₀ to the DOT1L IC₅₀ is betweenabout 10 and about 50. In certain embodiments, the invention relates toany one of the aforementioned compounds, wherein the compound inhibitsboth DOT1L and EHMT2; the compound has a DOT1L IC₅₀ of between about0.001 μM and about 10 μM; and the ratio of the EHMT2 IC₅₀ to the DOT1LIC₅₀ is between about 50 and about 100. In certain embodiments, theinvention relates to any one of the aforementioned compounds, whereinthe compound inhibits both DOT1L and EHMT2; the compound has a DOT1LIC₅₀ of between about 0.001 μM and about 10 μM; and the ratio of theEHMT2 IC₅₀ to the DOT1L IC₅₀ is between about 100 and about 1,000.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein the compound inhibits both DOT1L andCARM1; the compound has a DOT1L IC₅₀ of between about 0.001 μM and about10 μM; and the ratio of the CARM1 IC₅₀ to the DOT1L IC₅₀ is betweenabout 10 and about 50. In certain embodiments, the invention relates toany one of the aforementioned compounds, wherein the compound inhibitsboth DOT1L and CARM1; the compound has a DOT1L IC₅₀ of between about0.001 μM and about 10 μM; and the ratio of the CARM1 IC₅₀ to the DOT1LIC₅₀ is between about 50 and about 100. In certain embodiments, theinvention relates to any one of the aforementioned compounds, whereinthe compound inhibits both DOT1L and CARM1; the compound has a DOT1LIC₅₀ of between about 0.001 μM and about 10 μM; and the ratio of theCARM1 IC₅₀ to the DOT1L IC₅₀ is between about 100 and about 1,000.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein the compound inhibits both DOT1L andPRMT5; the compound has a DOT1L IC₅₀ of between about 0.001 μM and about10 μM; and the ratio of the PRMT5 IC₅₀ to the DOT1L IC₅₀ is betweenabout 10 and about 50. In certain embodiments, the invention relates toany one of the aforementioned compounds, wherein the compound inhibitsboth DOT1L and PRMT5; the compound has a DOT1L IC₅₀ of between about0.001 μM and about 10 μM; and the ratio of the PRMT5 IC₅₀ to the DOT1LIC₅₀ is between about 50 and about 100. In certain embodiments, theinvention relates to any one of the aforementioned compounds, whereinthe compound inhibits both DOT1L and PRMT5; the compound has a DOT1LIC₅₀ of between about 0.001 μM and about 10 μM; and the ratio of thePRMT5 IC₅₀ to the DOT1L IC₅₀ is between about 100 and about 1,000.

Many of the compounds of the invention may be provided as salts withpharmaceutically compatible counterions (i.e., pharmaceuticallyacceptable salts). A “pharmaceutically acceptable salt” means anynon-toxic salt that, upon administration to a recipient, is capable ofproviding, either directly or indirectly, a compound or a prodrug of acompound of this invention. A “pharmaceutically acceptable counterion”is an ionic portion of a salt that is not toxic when released from thesalt upon administration to a subject. Pharmaceutically compatible saltsmay be formed with many acids, including but not limited tohydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinicacids. Salts tend to be more soluble in water or other protic solventsthan their corresponding free base forms. The present invention includessuch salts.

Pharmaceutically acceptable acid addition salts include those formedwith mineral acids such as hydrochloric acid and hydrobromic acid, andalso those formed with organic acids such as maleic acid. For example,acids commonly employed to form pharmaceutically acceptable saltsinclude inorganic acids such as hydrogen bisulfide, hydrochloric,hydrobromic, hydroiodic, sulfuric and phosphoric acid, as well asorganic acids such as para-toluenesulfonic, salicylic, tartaric,bitartaric, ascorbic, maleic, besylic, fumaric, gluconic, glucuronic,formic, glutamic, methanesulfonic, ethanesulfonic, benzenesulfonic,lactic, oxalic, para-bromophenylsulfonic, carbonic, succinic, citric,benzoic and acetic acid, and related inorganic and organic acids. Suchpharmaceutically acceptable salts thus include sulfate, pyrosulfate,bisulfate, sulfite, bisulfate, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide,iodide, acetate, propionate, decanoate, caprylate, acrylate, formate,isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate,succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate,hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate,terephthalate, sulfonate, xylenesulfonate, phenylacetate,phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate,glycolate, maleate, tartrate, methanesulfonate, propanesulfonate,naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate and thelike.

Suitable bases for forming pharmaceutically acceptable salts with acidicfunctional groups include, but are not limited to, hydroxides of alkalimetals such as sodium, potassium, and lithium; hydroxides of alkalineearth metal such as calcium and magnesium; hydroxides of other metals,such as aluminum and zinc; ammonia, and organic amines, such asunsubstituted or hydroxy-substituted mono-, di-, or trialkylamines;dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine;diethylamine; triethylamine; mono-, bis-, or tris-(2-hydroxy-lower alkylamines), such as mono-, bis-, or tris-(2-hydroxyethyl)amine,2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine, N,N-dialkyl-N-(hydroxy alkyl)-amines, such asN,N-dimethyl-N-(2-hydroxyethyl)amine, or tri-(2-hydroxyethyl)amine;N-methyl-D-glucamine; and amino acids such as arginine, lysine, and thelike.

Certain compounds of the invention and their salts may exist in morethan one crystalline form (i.e., polymorph); the present inventionincludes each of the crystal forms and mixtures thereof.

Certain compounds of the invention may contain one or more chiralcenters, and exist in different optically active forms. When compoundsof the invention contain one chiral center, the compounds exist in twoenantiomeric forms and the present invention includes both enantiomersand mixtures of enantiomers, such as racemic mixtures thereof. Theenantiomers may be resolved by methods known to those skilled in theart; for example, enantiomers may be resolved by formation ofdiastereoisomeric salts which may be separated, for example, bycrystallization; formation of diastereoisomeric derivatives or complexeswhich may be separated, for example, by crystallization, gas-liquid orliquid chromatography; selective reaction of one enantiomer with anenantiomer-specific reagent, for example, via enzymatic esterification;or gas-liquid or liquid chromatography in a chiral environment, forexample, on a chiral support; suitable include chiral supports (e.g.,silica with a bound chiral ligand) or in the presence of a chiralsolvent. Where the desired enantiomer is converted into another chemicalentity by one of the separation procedures described above, a furtherstep may be used to liberate the desired purified enantiomer.Alternatively, specific enantiomers may be synthesized by asymmetricsynthesis using optically active reagents, substrates, catalysts orsolvents, or by converting one enantiomer into the other by asymmetrictransformation.

When a compound of the invention contains more than one chiral center,it may exist in diastereoisomeric forms. The diastereoisomeric compoundsmay be separated by methods known to those skilled in the art (forexample, chromatography or crystallization) and the individualenantiomers may be separated as described above. The present inventionincludes the various diastereoisomers of compounds of the invention, andmixtures thereof. Compounds of the invention may exist in differenttautomeric forms or as different geometric isomers, and the presentinvention includes each tautomer and/or geometric isomer of compounds ofthe invention, and mixtures thereof. Compounds of the invention mayexist in zwitterionic form. The present invention includes eachzwitterionic form of compounds of the invention, and mixtures thereof.

As used herein the term “prodrug” refers to an agent which is convertedinto the parent drug in vivo by some physiological chemical process(e.g., a prodrug on being brought to the physiological pH is convertedto the desired drug form). Prodrugs are often useful because, in somesituations. they may be easier to administer than the parent drug. Theymay, for instance, be bioavailable by oral administration whereas theparent drug is not. The prodrug may also have improved solubility inpharmacological compositions over the parent drug. An example, withoutlimitation, of a prodrug would be a compound of the present inventionwherein it is administered as an ester (the “prodrug”) to facilitatetransmittal across a cell membrane where water solubility is notbeneficial, but then it is metabolically hydrolyzed to the carboxylicacid once inside the cell where water solubility is beneficial. Prodrugshave many useful properties. For example, a prodrug may be more watersoluble than the ultimate drug, thereby facilitating intravenousadministration of the drug. A prodrug may also have a higher level oforal bioavailability than the ultimate drug. After administration, theprodrug is enzymatically or chemically cleaved to deliver the ultimatedrug in the blood or tissue.

Exemplary prodrugs release an amine of a compound of the inventionwherein the free hydrogen of an amine or alcohol is replaced by(C₁-C₆)alkanoyloxymethyl, 1-((C₁-C₆)alkanoyloxy)ethyl,1-methyl-1-((C₁-C₆)alkanoyloxy)ethyl, (C₁-C₆)alkoxycarbonyl-oxymethyl,N—(C₁-C₆)alkoxycarbonylamino-methyl, succinoyl, (C₁-C₆)alkanoyl,α-amino(C₁-C₄)alkanoyl, arylactyl and α-aminoacyl, orα-aminoacyl-α-aminoacyl wherein said α-aminoacyl moieties areindependently any of the naturally occurring L-amino acids found inproteins, —P(O)(OH)₂, —P(O)(O(C₁-C₆)alkyl)₂ or glycosyl (the radicalresulting from detachment of the hydroxyl of the hemiacetal of acarbohydrate).

Other exemplary prodrugs upon cleavage release a corresponding freeacid, and such hydrolyzable ester-forming residues of the compounds ofthe invention include but are not limited to carboxylic acidsubstituents (e.g., —(CH₂)C(O)OH or a moiety that contains a carboxylicacid) wherein the free hydrogen is replaced by (C₁-C₄)alkyl,(C₂-C₁₂)alkanoyloxymethyl, (C₄-C₉)1-(alkanoyloxy)ethyl,1-methyl-1-(alkanoyloxy)-ethyl having from 5 to 10 carbon atoms,alkoxycarbonyloxymethyl having from 3 to 6 carbon atoms,1-(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms,1-methyl-1-(alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms,N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms,1-(N-(alkoxycarbonyl)amino)ethyl having from 4 to 10 carbon atoms,3-phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl,di-N,N—(C₁-C₂)alkylamino(C₂-C₃)alkyl (such as β-dimethylaminoethyl),carbamoyl-(C₁-C₂)alkyl, N,N-di(C₁-C₂)-alkylcarbamoyl-(C₁-C₂)alkyl andpiperidino-, pyrrolidino- or morpholino(C₂-C₃)alkyl.

General Synthetic Schemes

The following describes the general synthetic procedures shown in FIGS.1 and 2. Unless noted otherwise, none of the specific conditions andreagents noted in the following is to be construed as limiting the scopeof the instant invention and are provided for illustrative purposesonly. All of the general procedures have been successfully performed andexemplifications of each general procedure are also provided.

FIG. 1 shows a general route for preparing compounds of formula viii,where R^(11a) is defined in the claim set. Conversion of vi to vii isaccomplished by treating with the appropriate isocyanate in a solvent,such as dimethylformamide. Treatment of the resultant urea vii with aprotic acid, such as trifluoroacetic acid, in the presence of water,will produce compounds of formula viii.

FIG. 2 shows a general route for the preparation of compounds of formulaxiii, where R, R′, R^(11a) and R^(11b) is defined in the claim set.Conversion of ix to xviii may be accomplished by treatment with theappropriate oxidizing agent, such as IBX, in the appropriate solvent,such as ethyl acetate. A reductive amination between xviii and x may beaccomplished by combining the two reagents in a standard solvent, suchas methanol, with a suitable catalyst, such as acetic acid, and anappropriate reductant, such as sodium cyanoborohydride. Treatment ofcompounds of formula xii under the conditions of method F will result incompounds of formula xiii.

In general, it may be convenient or desirable to prepare, purify, and/orhandle any of the compounds in FIGS. 1 and 2 in a chemically protectedform. The term “chemically protected form,” as used herein, pertains toa compound in which one or more reactive functional groups are protectedfrom undesirable chemical reactions (i.e., they have been modified witha protecting group).

By protecting a reactive functional group, reactions involving otherunprotected reactive functional groups can be performed withoutaffecting the protected group; the protecting group may be removed,usually in a subsequent step, without substantially affecting theremainder of the molecule. See, for example, Protective Groups inOrganic Synthesis (T. Green and P. Wuts, Wiley, 1991), and ProtectiveGroups in Organic Synthesis (T. Green and P. Wuts; 3rd Edition; JohnWiley and Sons, 1999).

For example, a hydroxy group may be protected as an ether (—OR) or anester (—OC(═O)R), for example, as: a t-butyl ether; a benzyl, benzhydryl(diphenylmethyl), or trityl (triphenylmethyl)ether; a trimethylsilyl ort-butyldimethylsilyl ether; or an acetyl ester (—OC(═O)CH₃,—OAc).

For example, an aldehyde or ketone group may be protected as an acetalor ketal, respectively, in which the carbonyl group (C(═O)) is convertedto a diether (C(OR)₂), by reaction with, for example, a primary alcohol.The aldehyde or ketone group is readily regenerated by hydrolysis usinga large excess of water in the presence of acid.

For example, an amine group may be protected, for example, as an amide(—NRC(═O)R) or a urethane (—NRC(═O)OR), for example, as: a methyl amide(—NHC(═O)CH₃); a benzyloxy amide (—NHC(═O)OCH₂C₆H₅NHCbz); as a t-butoxyamide (—NHC═(═O)OC(CH₃)₃, —NHBoc); a 2-biphenyl-2-propoxy amide(—NHC(═O)OC(CH₃)₂C₆H₄C₆H₅NHBoc), as a 9-fluorenylmethoxy amide(—NHFmoc), as a 6-nitroveratryloxy amide (—NHNvoc), as a2-trimethylsilylethyloxy amide (—NHTeoc), as a 2,2,2-trichloroethyloxyamide (—NHTroc), as an allyloxy amide (—NHAlloc), as a2-(phenylsulfonyl)ethyloxy amide (—NHPsec); or, in suitable cases (e.g.,cyclic amines), as a nitroxide radical.

For example, a carboxylic acid group may be protected as an ester or anamide, for example, as: a benzyl ester; a t-butyl ester; a methyl ester;or a methyl amide.

For example, a thiol group may be protected as a thioether (—SR), forexample, as: a benzyl thioether; or an acetamidomethyl ether(—SCH₂NHC(═O)CH₃).

For example, a benzimidazole group may be protected with a SEM or benzylprotecting group

“Tautomer” is one of two or more structural isomers that exist inequilibrium and which readily convert from one isomeric form to another.This conversion results in the formal migration of a hydrogen atomaccompanied by a switch of adjacent conjugated double bonds. Tautomersexist as a mixture of a tautomeric set in solution. In solutions wheretautomerization is possible, a chemical equilibrium of the tautomerswill be reached. The exact ratio of the tautomers depends on severalfactors, including temperature, solvent and pH. The concept of tautomersthat are interconvertable by tautomerizations is called tautomerism. Ofthe various types of tautomerism that are possible, two are commonlyobserved. In keto-enol tautomerism a simultaneous shift of electrons anda hydrogen atom occurs. Ring-chain tautomerism arises as a result of thealdehyde group (—CHO) in a sugar chain molecule reacting with one of thehydroxy groups (—OH) in the same molecule to give it a cyclic(ring-shaped) form as exhibited by glucose. Common tautomeric pairsinclude: ketone-enol, amide-nitrile, lactam-lactim, amide-imidic acidtautomerism in heterocyclic rings (e.g., in nucleobases such as guanine,thymine and cytosine), amine-enamine and enamine-enamine.

Benzimidazoles also exhibit tautomerism: when the benzimidazole containsone or more substituents in the 4-, 5-, 6- or 7-positions, thepossibility of different isomers arises. For example,2,5-dimethyl-1H-benzo[d]imidazole can exist in equilibrium with itsisomer 2,6-dimethyl-1H-benzo[d]imidazole via tautomerization.

It is to be understood that the compounds of the present invention maybe depicted as different tautomers. It should also be understood thatwhen compounds have tautomeric forms, all tautomeric forms are intendedto be included in the scope of the present invention, and the specificnaming convention used for a particular compound does not exclude anytautomer form.

Pharmaceutical Compositions

One or more compounds of the invention can be administered to a humanpatient by themselves or in pharmaceutical compositions where they aremixed with suitable carriers or excipient(s) at doses to treat orameliorate a disease or condition as described herein. Mixtures of thesecompounds can also be administered to the patient as a simple mixture orin suitable formulated pharmaceutical compositions. For example, oneaspect of the invention relates to pharmaceutical composition comprisinga therapeutically effective dose of a compound of formula I, or apharmaceutically acceptable salt, hydrate, enantiomer or stereoisomerthereof; and a pharmaceutically acceptable diluent or carrier; whereinthe compound of formula I is represented by

wherein independently for each occurrence,

R¹ is hydrogen, alkyl, cycloalkyl, alkylcycloalkyl, alkylaryl,haloalkyl, formyl, heterocyclyl, heterocyclylalkyl,

or (C₂-C₄)alkyl substituted with

except that when X is

R¹ is not

R¹⁰ is hydrogen or alkyl;

R^(11a) is hydrogen, alkyl, or alkyl-cycloalkyl;

R^(11b) is hydrogen or alkyl; or taken together with R^(11a) and thenitrogen to which it is attached forms a 4- to 8-membered heterocyclylcomprising 0 or 1 additional heteroatoms;

R¹³ is hydrogen, alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl orsilyl;

R¹⁴ is hydrogen, alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R¹⁵ is alkyl, cycloalkyl or cycloalkylalkyl;

R²⁰ is hydrogen, alkyl, cycloalkyl or cycloalkylalkyl;

A is

R² is

Y is —NH—, —N(alkyl)-, —O—, or —CR⁶ ₂—;

R^(22a) is aryl, heteroaryl, aralkyl, heteroaralkyl, fused bicyclyl,biaryl, aryloxyaryl, heteroaryloxyaryl, aryloxyheteroaryl orheteroaryloxyheteroaryl;

R^(22b) is hydrogen or alkyl;

R²⁴ is hydrogen or alkyl;

R^(25a), R^(25b), R^(25c), and R^(25d) independently are -M₂-T₂, inwhich M₂ is a bond, SO₂, SO, S, CO, CO₂, O, O—C₁-C₄ alkyl linker, C₁-C₄alkyl linker, NH, or N(R_(t)), R_(t) being C₁-C₆ alkyl, and T₂ is H,halo, or R_(S4), R_(S4) being C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 8-membered heterocycloalkyl, or 5 to10-membered heteroaryl, and each of O—C₁-C₄ alkyl linker, C₁-C₄ alkyllinker, R_(t), and R_(S4) being optionally substituted with one or moresubstituents selected from the group consisting of halo, hydroxyl,carboxyl, cyano, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 6-membered heterocycloalkyl, and 5 to6-membered heteroaryl;

R³ is hydrogen, halogen, hydroxy, alkyloxy, aralkyloxy, alkylcarbonyloxyor silyloxy;

R⁴ is hydrogen, halogen, hydroxy, alkyloxy, aralkyloxy, alkylcarbonyloxyor silyloxy;

R⁴¹ is hydrogen, alkyl or alkynyl;

Z is hydrogen or

R^(5a) is hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aryl, heteroaryl, biaryl, alkenylalkyl, alkynylalkyl,carbocyclylalkyl, heterocyclylalkyl, aralkyl, heteroaralkyl,alkylcarbonylaminoalkyl, arylcarbonylaminoalkyl,aralkylcarbonylaminoalkyl, arylsulfonylaminoalkyl, alkylthioalkyl,aralkylthioalkyl or heteroaralkylthioalkyl; or alkyl substituted with 1,2 or 3 substituents independently selected from the group consisting ofhydroxy, halo, carboxy, alkyoxy, aryloxy, aralkyloxy, nitro, amino,amido, aryl and heteroaryl:

R^(5b) is hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aryl, heteroaryl, biaryl, alkenylalkyl, alkynylalkyl,carbocyclylalkyl, heterocyclylalkyl, aralkyl, heteroaralkyl,alkylcarbonylaminoalkyl, arylcarbonylaminoalkyl,aralkylcarbonylaminoalkyl, arylsulfonylaminoalkyl, alkylthioalkyl,aralkylthioalkyl or heteroaralkylthioalkyl; or alkyl substituted with 1,2 or 3 substituents independently selected from the group consisting ofhydroxy, halo, carboxy, alkyoxy, aryloxy, aralkyloxy, nitro, amino,amido, aryl and heteroaryl; or taken together with R^(5a) and thenitrogen to which it is attached forms a 4- to 8-membered heterocyclylcomprising 0 or 1 additional heteroatoms;

R⁶ is hydrogen, alkyl or halo; or two geminal R⁶ taken together areethylene, propylene or butylene;

R^(7a) is hydrogen, lower alkyl, lower haloalkyl, cyano, halo, loweralkoxy, or C₃-C₅ cycloalkyl, optionally substituted with 1, 2 or 3substituents independently selected from the group consisting of cyano,lower alkoxy and halo;

R^(7b) is hydrogen, lower alkyl, lower haloalkyl, cyano, halo, loweralkoxy, or C₃-C₅ cycloalkyl, optionally substituted with 1, 2 or 3substituents independently selected from the group consisting of cyano,lower alkoxy and halo; and

R^(7c) is hydrogen, lower alkyl, lower haloalkyl, cyano, halo, loweralkoxy, or C₃-C₅ cycloalkyl, optionally substituted with 1, 2 or 3substituents independently selected from the group consisting of cyano,lower alkoxy and halo.

Techniques for formulation and administration of the compounds of theinstant application may be found in references well known to one ofordinary skill in the art, such as “Remington's PharmaceuticalSciences,” Mack Publishing Co., Easton, Pa., latest edition.

Suitable routes of administration may, for example, include oral,eyedrop, rectal, transmucosal, topical, or intestinal administration;parenteral delivery, including intramuscular, subcutaneous,intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections.

Alternatively, one may administer a compound in a local rather than asystemic manner, for example, via injection of the compound directlyinto an edematous site, often in a depot or sustained releaseformulation.

Furthermore, one may administer a compound in a targeted drug deliverysystem, for example, in a liposome coated with endothelial-cell-specificantibody.

The pharmaceutical compositions of the present invention may bemanufactured, e.g., by conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping orlyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in a conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks' solution, Ringer's solution, or physiological saline buffer. Fortransmucosal administration, penetrants are used in the formulationappropriate to the barrier to be permeated. Such penetrants aregenerally known in the art.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained by combining the active compound with a solidexcipient, optionally grinding a resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients include fillers suchas sugars, including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebuliser, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds can be formulated for parenteral administration byinjection, e.g., bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form forreconstitution before use with a suitable vehicle, e.g., sterilepyrogen-free water.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example, subcutaneously orintramuscularly or by intramuscular injection). Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives (for example, as asparingly soluble salt).

Alternatively, other delivery systems for hydrophobic pharmaceuticalcompounds may be employed. Liposomes and emulsions are examples ofdelivery vehicles or carriers for hydrophobic drugs. Certain organicsolvents such as dimethylsulfoxide also may be employed. Additionally,the compounds may be delivered using a sustained-release system, such assemi-permeable matrices of solid hydrophobic polymers containing thetherapeutic agent. Various sustained-release materials have beenestablished and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days. Depending onthe chemical nature and the biological stability of the therapeuticreagent, additional strategies for protein stabilization may beemployed.

The pharmaceutical compositions may also comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymers,such as polyethylene glycols.

Methods of Treatment

Provided herein are methods of treating or preventing conditions anddiseases the course of which can be influenced by modulating themethylation status of histones or other proteins, wherein saidmethylation status is mediated at least in part by the activity of DOT1.Modulation of the methylation status of histones can in turn influencethe level of expression of target genes activated by methylation, and/ortarget genes suppressed by methylation. For example, one aspect of theinvention relates to a method comprising administering to a subject inneed thereof a therapeutically effective amount of a compound of formulaI, or a pharmaceutically acceptable salt, hydrate, enantiomer orstereoisomer thereof; wherein the compound of formula I is representedby

wherein independently for each occurrence,

R¹ is hydrogen, alkyl, cycloalkyl, alkylcycloalkyl, alkylaryl,haloalkyl, formyl, heterocyclyl, heterocyclylalkyl,

or (C₂-C₄)alkyl substituted with

except that when X is

R¹ is not

R¹⁰ is hydrogen or alkyl;

R^(11a) is hydrogen, alkyl, or alkyl-cycloalkyl;

R^(11b) is hydrogen or alkyl; or taken together with R^(11a) and thenitrogen to which it is attached forms a 4- to 8-membered heterocyclylcomprising 0 or 1 additional heteroatoms;

R¹³ is hydrogen, alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl orsilyl;

R¹⁴ is hydrogen, alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R¹⁵ is alkyl, cycloalkyl or cycloalkylalkyl;

R²⁰ is hydrogen, alkyl, cycloalkyl or cycloalkylalkyl;

A is

R² is

Y is —NH—, —N(alkyl)-, —O—, or —CR⁶ ₂—;

R^(22a) is aryl, heteroaryl, aralkyl, heteroaralkyl, fused bicyclyl,biaryl, aryloxyaryl, heteroaryloxyaryl, aryloxyheteroaryl orheteroaryloxyheteroaryl;

R^(22b) is hydrogen or alkyl;

R²⁴ is hydrogen or alkyl;

R^(25a), R^(25b), R^(25c), and R^(25d) independently are -M₂-T₂, inwhich M₂ is a bond, SO₂, SO, S, CO, CO₂, O, O—C₁-C₄ alkyl linker, C₁-C₄alkyl linker, NH, or N(R_(t)), R_(t) being C₁-C₆ alkyl, and T₂ is H,halo, or R_(S4), R_(S4) being C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 8-membered heterocycloalkyl, or 5 to10-membered heteroaryl, and each of O—C₁-C₄ alkyl linker, C₁-C₄ alkyllinker, R_(t), and R_(S4) being optionally substituted with one or moresubstituents selected from the group consisting of halo, hydroxyl,carboxyl, cyano, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 6-membered heterocycloalkyl, and 5 to6-membered heteroaryl;

R³ is hydrogen, halogen, hydroxy, alkyloxy, aralkyloxy, alkylcarbonyloxyor silyloxy;

R⁴ is hydrogen, halogen, hydroxy, alkyloxy, aralkyloxy, alkylcarbonyloxyor silyloxy;

R⁴¹ is hydrogen, alkyl or alkynyl;

R^(5a) is hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aryl, heteroaryl, biaryl, alkenylalkyl, alkynylalkyl,carbocyclylalkyl, heterocyclylalkyl, aralkyl, heteroaralkyl,alkylcarbonylaminoalkyl, arylcarbonylaminoalkyl,aralkylcarbonylaminoalkyl, arylsulfonylaminoalkyl, alkylthioalkyl,aralkylthioalkyl or heteroaralkylthioalkyl; or alkyl substituted with 1,2 or 3 substituents independently selected from the group consisting ofhydroxy, halo, carboxy, alkyoxy, aryloxy, aralkyloxy, nitro, amino,amido, aryl and heteroaryl:

R^(5b) is hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aryl, heteroaryl, biaryl, alkenylalkyl, alkynylalkyl,carbocyclylalkyl, heterocyclylalkyl, aralkyl, heteroaralkyl,alkylcarbonylaminoalkyl, arylcarbonylaminoalkyl,aralkylcarbonylaminoalkyl, arylsulfonylaminoalkyl, alkylthioalkyl,aralkylthioalkyl or heteroaralkylthioalkyl; or alkyl substituted with 1,2 or 3 substituents independently selected from the group consisting ofhydroxy, halo, carboxy, alkyoxy, aryloxy, aralkyloxy, nitro, amino,amido, aryl and heteroaryl: or taken together with R^(5a) and thenitrogen to which it is attached forms a 4- to 8-membered heterocyclylcomprising 0 or 1 additional heteroatoms;

R⁶ is hydrogen, alkyl or halo; or two geminal R⁶ taken together areethylene, propylene or butylene;

R^(7a) is hydrogen, lower alkyl, lower haloalkyl, cyano, halo, loweralkoxy, or C₃-C₅ cycloalkyl, optionally substituted with 1, 2 or 3substituents independently selected from the group consisting of cyano,lower alkoxy and halo;

R^(7b) is hydrogen, lower alkyl, lower haloalkyl, cyano, halo, loweralkoxy, or C₃-C₅ cycloalkyl, optionally substituted with 1, 2 or 3substituents independently selected from the group consisting of cyano,lower alkoxy and halo; and

R^(7c) is hydrogen, lower alkyl, lower haloalkyl, cyano, halo, loweralkoxy, or C₃-C₅ cycloalkyl, optionally substituted with 1, 2 or 3substituents independently selected from the group consisting of cyano,lower alkoxy and halo.

In certain embodiments, the invention related to any one of theaforementioned methods, wherein Z is hydrogen.

In certain embodiments, the invention related to any one of theaforementioned methods, wherein Z is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein X is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein X is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein X is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein X is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein X is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein X is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein X is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R² is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R² is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R²⁴ is hydrogen or alkyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R²⁴ is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R^(25a) is hydrogen, alkyl, —O-alkyl,halogen, trifluoroalkyl, —O-trifluoromethyl, or —SO₂-trifluoromethyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R^(25b) is hydrogen, alkyl, halogen, ortrifluoroalkyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R^(25c) is hydrogen, alkyl, or halogen.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R^(25c) is hydrogen or halogen.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R² is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein Y is —NH— or —N(alkyl)-.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein Y is —NH—.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein Y is —N(CH₃)—.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein Y is —O—.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein Y is —CH₂—.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R^(22a) is aryl or aralkyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R^(22a) is substituted phenyl orsubstituted benzyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R^(22a) is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R^(22a) is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R^(22b) is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R^(22b) is methyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R¹ is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R¹ is alkyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R¹ is —CH₃, —CH₂CH₃, —CH₂CH(CH₃)₂ or—CH₂CH₂CH(CH₃)₂.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R¹ is C₃-C₇ cycloalkyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R¹ is cyclopropyl, cyclopropylmethyl,2-cyclopropylethyl, cyclobutyl, cyclobutylmethyl, 2-cyclobutylethyl,cyclopentyl, cyclopentylmethyl, or 2-cyclopentylethyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R¹ is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R¹ is —CH₂CF₃.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R¹ is —CH₂Ph.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R¹ is —C(═O)H.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R¹ is —C(═O)CH₃.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is heterocyclyl orheterocyclylalkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R¹ is

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R¹ is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R¹⁵ is alkyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R¹⁵ is cycloalkyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R¹⁵ is cycloalkylalkyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R¹ is (C₂-C₄)alkyl substituted with

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R¹ is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R^(11a) is hydrogen, alkyl, oralkyl-cycloalkyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R^(11a) is hydrogen, methyl, ori-propyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R¹ is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R¹³ is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R¹ is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein A is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein A is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein A is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein A is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein A is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein A is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein A is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein A is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein A is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein A is

and R⁶ is alkyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein A is

and R⁶ is methyl, ethyl or isopropyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R³ is hydroxyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R³ is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R⁴ is hydroxyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R⁴ is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R⁴¹ is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R⁴¹ is methyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R³ is hydroxyl; and R⁴ is hydroxyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R³ is hydroxyl; R⁴ is hydroxyl; and R⁴¹is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R³ is hydroxyl; R⁴ is hydroxyl; and R⁴¹is methyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R³ is hydrogen; and R⁴ is hydroxyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R³ is hydrogen; R⁴ is hydroxyl; and R⁴¹is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R³ is hydrogen; R⁴ is hydroxyl; and R⁴¹is methyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R³ is hydroxyl; and R⁴ is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein Z is hydrogen or

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein Z is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein Z is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R^(5a) is hydrogen, alkyl, carbocyclyl,heterocyclyl, aryl, heteroaryl, carbocyclylalkyl, heterocyclylalkyl,aralkyl, or heteroaralkyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R^(5a) is hydrogen, aralkyloxyalkyl,alkyl, aryl, aralkyl, aminoalkyl or hydroalkyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R^(5a) is —H, —CH₂CH₂OCH₂Ph, —CH₂CH₃,—CH(CH₃)₂, -Ph, —CH₂CH(CH₃), —CH₃, —CH₂Ph, —CH₂CH₂NH₂, —CH₂(cyclohexyl)or —CH₂CH₂OH.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R^(5b) is hydrogen, alkyl, carbocyclyl,heterocyclyl, aryl, heteroaryl, carbocyclylalkyl, heterocyclylalkyl,aralkyl, or heteroaralkyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R^(5b) is hydrogen, aralkyloxyalkyl,alkyl, aryl, aralkyl, aminoalkyl or hydroalkyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R^(5b) is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R^(5a) is —H, —CH₂CH₂OCH₂Ph, —CH₂CH₃,—CH(CH₃)₂, -Ph, —CH₂CH(CH₃), —CH₃, —CH₂Ph, —CH₂CH₂NH₂, —CH₂(cyclohexyl)or —CH₂CH₂OH; and R^(5b) is —H.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R^(7a) is hydrogen or lower alkyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R^(7a) is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R^(7b) is hydrogen or lower alkyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R^(7b) is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R^(7c) is hydrogen or lower alkyl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein R^(7c) is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the compound, or a pharmaceuticallyacceptable salt, hydrate, enantiomer or stereoisomer thereof, isselected from the group consisting of

Diseases such as cancers and neurological disease can be treated byadministration of modulators of protein (e.g., histone) methylation,e.g., modulators of histone methyltransferase, or histone demethylaseenzyme activity. Histone methylation has been reported to be involved inaberrant expression of certain genes in cancers, and in silencing ofneuronal genes in non-neuronal cells. Modulators described herein can beused to treat these diseases, i.e., to restore normal methylation statesof histones or other proteins to affected cells.

Based at least on the fact that increased histone methylation has beenfound to be associated with certain cancers, a method for treatingcancer in a subject comprises administering to the subject in needthereof a therapeutically effective amount of a compound that decreasesmethylation or restores methylation to roughly its level in counterpartnormal cells. It is important to note that disease-specific increase inmethylation can occur at chromatin in key genomic loci in the absence ofa global increase in cellular levels of histone or protein methylation.For example, it is possible for aberrant hypermethylation at keydisease-relevant genes to occur against a backdrop of global histone orprotein hypomethylation,

Modulators of methylation can be used for modulating cell proliferation,generally. For example, in some cases excessive proliferation may bereduced with agents that decrease methylation, whereas insufficientproliferation may be stimulated with agents that increase methylation.Accordingly, diseases that may be treated include hyperproliferativediseases, such as benign cell growth and malignant cell growth.

Exemplary cancers that may be treated include leukemias, e.g., acutelymphoid leukemia and myeloid leukemia, mixed lineage leukemia andcarcinomas, such as colorectal carcinoma and hepatocarcinoma. Othercancers include Acute Lymphoblastic Leukemia; Acute LymphoblasticLeukemia; Acute Myeloid Leukemia; Acute Myeloid Leukemia; AdrenocorticalCarcinoma Adrenocortical Carcinoma; AIDS-Related Cancers; AIDS-RelatedLymphoma; Anal Cancer: Astrocytoma, Childhood Cerebellar; Astrocytoma,Childhood Cerebral; Basal Cell Carcinoma, see Skin Cancer(non-Melanoma); Bile Duct Cancer, Extrahepatic; Bladder Cancer; BladderCancer; Bone Cancer, osteosarcoma/Malignant Fibrous Histiocytoma; BrainStem Glioma; Brain Tumor; Brain Tumor, Brain Stem Glioma; Brain Tumor,Cerebellar Astrocytoma; Brain Tumor, Cerebral Astrocytoma/MalignantGlioma; Brain Tumor. Ependymoma; Brain Tumor, Medulloblastoma; BrainTumor, Supratentoria I Primitive Neuroectodermal Tumors; Brain Tumor,Visual Pathway and Hypothalamic Glioma; Brain Tumor; Breast Cancer;Breast Cancer and Pregnancy; Breast Cancer; Breast Cancer, Male;Bronchial Adenomas/Carcinoids; Burkitt's Lymphoma; Carcinoid Tumor;Carcinoid Tumor, Gastrointestinal; Carcinoma of Unknown Primary; CentralNervous System Lymphoma, Primary; Cerebellar Astrocytoma; CerebralAstrocytoma/Malignant Glioma; Cervical Cancer; Childhood Cancers;Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia; ChronicMyeloproliferative Disorders; Colon Cancer; Colorectal Cancer; CutaneousT-Cell Lymphoma, see Mycosis Fungoides and Sezary Syndrome; EndometrialCancer; Ependymoma; Esophageal Cancer; Esophageal Cancer; Ewing's Familyof Tumors; Extracranial Germ Cell Tumor; Extragonadal Germ Cell Tumor;Extrahepatic Bile Duct Cancer; Eye Cancer, Intraocular Melanoma; EyeCancer, Retinoblastoma; Gallbladder Cancer; Gastric (Stomach) Cancer;Gastric (Stomach) Cancer; Gastrointestinal Carcinoid Tumor; Germ CellTumor, Extracranial; Germ Cell Tumor, Extragonadal; Germ Cell Tumor,Ovarian; Gestational Trophoblastic Tumor; Glioma; Glioma, ChildhoodBrain Stem; Glioma, Childhood Cerebral Astrocytoma; Glioma, ChildhoodVisual Pathway and Hypothalamic; Hairy Cell Leukemia; Head and NeckCancer; Hepatocellular (Liver) Cancer, Adult (Primary); Hepatocellular(Liver) Cancer, Childhood (Primary); Hodgkin's Lymphoma; Hodgkin'sLymphoma; Hodgkin's Lymphoma During Pregnancy; Hypopharyngeal Cancer;Hypothalamic and Visual Pathway Glioma; Intraocular Melanoma; Islet CellCarcinoma (Endocrine Pancreas); Kaposi's Sarcoma; Kidney (Renal Cell)Cancer; Kidney Cancer; Laryngeal Cancer; Laryngeal Cancer; Leukemia,Acute Lymphoblastic; Leukemia, Acute Lymphoblastic; Leukemia, AcuteMyeloid; Leukemia, Acute Myeloid; Leukemia, Chronic Lymphocytic;Leukemia; Chronic Myelogenous; Leukemia, Hairy Cell; Lip and Oral CavityCancer; Liver Cancer, Adult (Primary); Liver Cancer, Childhood(Primary); Lung Cancer, Non-Small Cell; Lung Cancer, Small Cell;Lymphoma, AIDS-Related; Lymphoma, Burkitt's; Lymphoma, Cutaneous T-Cell,see Mycosis Fungoides and Sezary Syndrome; Lymphoma, Hodgkin's;Lymphoma, Hodgkin's; Lymphoma, Hodgkin's During Pregnancy; Lymphoma,Non-Hodgkin's; Lymphoma, Non-Hodgkin's; Lymphoma, Non-Hodgkin's DuringPregnancy; Lymphoma, Primary Central Nervous System; Macroglobulinemia,Waldenstrom's; Malignant Fibrous Histiocytoma of Bone/Osteosarcoma;Medulloblastoma; Melanoma; Melanoma, Intraocular (Eye); Merkel CellCarcinoma; Mesothelioma, Adult Malignant; Mesothelioma; MetastaticSquamous Neck Cancer with Occult Primary; Multiple Endocrine NeoplasiaSyndrome; Multiple Myeloma/Plasma Cell Neoplasm' Mycosis Fungoides;Myelodysplastic Syndromes; Myelodysplastic/Myeloproliferative Diseases;Myelogenous Leukemia, Chronic; Myeloid Leukemia, Adult Acute; MyeloidLeukemia, Childhood Acute; Myeloma, Multiple; MyeloproliferativeDisorders, Chronic; Nasal Cavity and Paranasal Sinus Cancer;Nasopharyngeal Cancer; Nasopharyngeal Cancer; Neuroblastoma;Non-Hodgkin's Lymphoma; Non-Hodgkin's Lymphoma; Non-Hodgkin's LymphomaDuring Pregnancy; Non-Small Cell Lung Cancer; Oral Cancer; Oral CavityCancer, Lip and; Oropharyngeal Cancer; Osteosarcoma/Malignant FibrousHistiocytoma of Bone; Ovarian Cancer; Ovarian Epithelial Cancer; OvarianGerm Cell Tumor; Ovarian Low Malignant Potential Tumor; PancreaticCancer; Pancreatic Cancer; Pancreatic Cancer, Islet Cell; ParanasalSinus and Nasal Cavity Cancer; Parathyroid Cancer; Penile Cancer;Pheochromocytoma; Pineoblastoma and Supratentorial PrimitiveNeuroectodermal Tumors; Pituitary Tumor; Plasma Cell Neoplasm/MultipleMyeloma; Pleuropulmonary Blastoma; Pregnancy and Breast Cancer;Pregnancy and Hodgkin's Lymphoma; Pregnancy and Non-Hodgkin's Lymphoma;Primary Central Nervous System Lymphoma; Prostate Cancer; Rectal Cancer;Renal Cell (Kidney) Cancer; Renal Cell (Kidney) Cancer; Renal Pelvis andUreter, Transitional Cell Cancer; Retinoblastoma; Rhabdomyosarcoma;Salivary Gland Cancer; Salivary Gland Cancer; Sarcoma, Ewing's Family ofTumors; Sarcoma, Kaposi's; Sarcoma, Soft Tissue; Sarcoma, Soft Tissue;Sarcoma, Uterine; Sezary Syndrome; Skin Cancer (non-Melanoma); SkinCancer; Skin Cancer (Melanoma); Skin Carcinoma, Merkel Cell; Small CellLung Cancer; Small Intestine Cancer; Soft Tissue Sarcoma; Soft TissueSarcoma; Squamous Cell Carcinoma, see Skin Cancer (non-Melanoma);Squamous Neck Cancer with Occult Primary, Metastatic; Stomach (Gastric)Cancer; Stomach (Gastric) Cancer; Supratentorial PrimitiveNeuroectodermal Tumors; T-Cell Lymphoma, Cutaneous, see MycosisFungoides and Sezary Syndrome; Testicular Cancer; Thymoma; Thymoma andThymic Carcinoma; Thyroid Cancer; Thyroid Cancer; Transitional CellCancer of the Renal Pelvis and Ureter; Trophoblastic Tumor, Gestational;Unknown Primary Site, Carcinoma of; Unknown Primary Site, Cancer of;Unusual Cancers of Childhood; Ureter and Renal Pelvis, Transitional CellCancer; Urethral Cancer; Uterine Cancer, Endometrial; Uterine Sarcoma;Vaginal Cancer; Visual Pathway and Hypothalamic Glioma; Vulvar Cancer;Waldenstrom's Macroglobulinemia; Wilms' Tumor; and Women's Cancers.

Neurologic diseases that may be treated include epilepsy, schizophrenia,bipolar disorder or other psychological and/or psychiatric disorders,neuropathies, skeletal muscle atrophy, and neurodegenerative diseases,e.g., a neurodegenerative disease. Exemplary neurodegenerative diseasesinclude: Alzheimer's, Amyotrophic Lateral Sclerosis (ALS), andParkinson's disease. Another class of neurodegenerative diseasesincludes diseases caused at least in part by aggregation ofpoly-glutamine. Diseases of this class include: Huntington's Diseases,Spinalbulbar Muscular Atrophy (SB MA or Kennedy's Disease)Dentatorubropallidoluysian Atrophy (DRPLA), Spinocerebellar Ataxia 1(SCA1), Spinocerebellar Ataxia 2 (SCA2), Machado-Joseph Disease (MJD;SCA3), Spinocerebellar Ataxia 6 (SCA6), Spinocerebellar Ataxia 7 (SCAT),and Spinocerebellar Ataxia 12 (SCA12).

Any other disease in which epigenetic methylation, which is mediated byDOT1, plays a role may be treatable or preventable using compounds andmethods described herein.

Combination Therapy

In one aspect of the invention, a compound of the invention, or apharmaceutically acceptable salt thereof, can be used in combinationwith another therapeutic agent to treat diseases such cancer and/orneurological disorders. For example, the additional agent can be atherapeutic agent that is art-recognized as being useful to treat thedisease or condition being treated by the compound of the presentinvention. The additional agent also can be an agent that imparts abeneficial attribute to the therapeutic composition (e.g., an agent thataffects the viscosity of the composition).

The combination therapy contemplated by the invention includes, forexample, administration of a compound of the invention, or apharmaceutically acceptable salt thereof, and additional agent(s) in asingle pharmaceutical formulation as well as administration of acompound of the invention, or a pharmaceutically acceptable saltthereof, and additional agent(s) in separate pharmaceuticalformulations. In other words, co-administration shall mean theadministration of at least two agents to a subject so as to provide thebeneficial effects of the combination of both agents. For example, theagents may be administered simultaneously or sequentially over a periodof time.

The agents set forth below are for illustrative purposes and notintended to be limited. The combinations, which are part of thisinvention, can be the compounds of the present invention and at leastone additional agent selected from the lists below. The combination canalso include more than one additional agent, e.g., two or threeadditional agents if the combination is such that the formed compositioncan perform its intended function.

For example, one aspect of the invention relates to the use of acompound of the invention (e.g., those of formula I) in combination withanother anticancer agent, e.g., a compound that effects histonemodifications, such as an HDAC inhibitor, for the treatment of cancerand/or a neurological disorder. In certain embodiments, the otheranticancer agent is selected from the group consisting ofchemotherapetics (such as 2CdA, 5-FU, 6-Mercaptopurine, 6-TG, Abraxane™,Accutane®, Actinomycin-D, Adriamycin®, Alimta®, all-trans retinoic acid,amethopterin, Ara-C, Azacitadine, BCNU, Blenoxane®, Camptosar®, CeeNU®,Clofarabine, Clolar™, Cytoxan®, daunorubicin hydrochloride, DaunoXome®,Dacogen®, DIC, Doxil®, Ellence®, Eloxatin®, Emcyt®, etoposide phosphate,Fludara®, FUDR®, Gemzar®, Gleevec®, hexamethylmelamine, Hycamtin®,Hydrea®, Idamycin®, Ifex®, ixabepilone, Ixempra®, L-asparaginase,Leukeran®, liposomal Ara-C, L-PAM, Lysodren, Matulane®, mithracin,Mitomycin-C, Myleran®, Navelbine®, Neutrexin®, nilotinib, Nipent®,Nitrogen Mustard, Novantrone®, Oncaspar®, Panretin®, Paraplatin®,Platinol®, prolifeprospan 20 with carmustine implant, Sandostatin®,Targretin®, Tasigna®, Taxotere®, Temodar®, TESPA, Trisenox®, Valstar®,Velban®, Vidaza™, vincristine sulfate, VM 26, Xeloda® and Zanosar®)biologics (such as Alpha Interferon, Bacillus Calmette-Guerin, Bexxar®,Campath®, Ergamisol®, Erlotinib, Herceptin®, Interleukin-2, Iressa®,lenalidomide, Mylotarg®, Ontak®, Pegasys®, Revlimid®, Rituxan®,Tarceva™, Thalomid®, Tykerb®, Velcade® and Zevalin™) corticosteroids,(such as dexamethasone sodium phosphate, DeltaSone® and Delta-Cortef®),hormonal therapies (such as Arimidex®, Aromasin®, Casodex®, Cytadren®,Eligard®, Eulexin®, Evista®, Faslodex®, Femara®, Halotestin®, Megace®,Nilandron®, Nolvadex®, Plenaxis™ and Zoladex®) and radiopharmaceuticals(such as Iodotope®, Metastron®, Phosphocol® and Samarium SM-153).

Dosage

As used herein, a “therapeutically effective amount” or “therapeuticallyeffective dose” is an amount of a compound of the invention or acombination of two or more such compounds, which inhibits, totally orpartially, the progression of the condition or alleviates, at leastpartially, one or more symptoms of the condition. A therapeuticallyeffective amount can also be an amount which is prophylacticallyeffective. The amount which is therapeutically effective will dependupon the patient's size and gender, the condition to be treated, theseverity of the condition and the result sought. For a given patient, atherapeutically effective amount may be determined by methods known tothose of skill in the art.

A therapeutically effective dose refers to that amount of the compoundthat results in amelioration of symptoms in a patient. Toxicity andtherapeutic efficacy of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the maximum tolerated dose (MTD) and the ED₅₀(effective dose for 50% maximal response). The dose ratio between toxicand therapeutic effects is the therapeutic index and it can be expressedas the ratio between MTD and ED₅₀. The data obtained from these cellculture assays and animal studies can be used in formulating a range ofdosage for use in humans. Dosage may also be guided by monitoringcompound effects on pharmacodynamic markers of enzyme inhibition (e.g.,histone methylation or target gene expression) in diseased or surrogatetissue. Cell culture or animal experiments can be used to determine therelationship between doses required for changes in pharmacodynamicmarkers and doses required for therapeutic efficacy can be determined incell culture or animal experiments or early stage clinical trials. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. In thetreatment of crises, the administration of an acute bolus or an infusionapproaching the MTD may be required to obtain a rapid response.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety which are sufficient to maintain themethyltransferase modulating effects, or minimal effective concentration(MEC) for the required period of time to achieve therapeutic efficacy.The MEC will vary for each compound but can be estimated from in vitrodata and animal experiments. Dosages necessary to achieve the MEC willdepend on individual characteristics and route of administration.However, HPLC assays or bioassays can be used to determine plasmaconcentrations.

Dosage intervals can also be determined using the MEC value. In certainembodiments, compounds should be administered using a regimen whichmaintains plasma levels above the MEC for 10-90% of the time, preferablybetween 30-90% and most preferably between 50-90% until the desiredamelioration of symptoms is achieved. In other embodiments, differentMEC plasma levels will be maintained for differing amounts of time. Incases of local administration or selective uptake, the effective localconcentration of the drug may not be related to plasma concentration.

One of skill in the art can select from a variety of administrationregimens and the amount of composition administered will, of course, bedependent on the subject being treated, on the subject's weight, theseverity of the affliction, the manner of administration and thejudgment of the prescribing physician.

Kits

The compounds and compositions of the invention (e.g., compounds andcompositions of formula I) may, if desired, be presented in a kit (e.g.,a pack or dispenser device) which may contain one or more unit dosageforms containing the active ingredient. The pack may for examplecomprise metal or plastic foil, such as a blister pack. The pack ordispenser device may be accompanied by instructions for administration.Compositions comprising a compound of the invention formulated in acompatible pharmaceutical carrier may also be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition. Instructions for use may also be provided.

Assessment of Activity of Compounds

DOT1L polypeptides and nucleic acids can be used to screen for compoundsthat bind to and/or modulate (e.g., increase or decrease) one or morebiological activities of DOT1L, including but not limited to H3K79HMTase activity, SAM binding activity, histone and/or nucleosome bindingactivity, AF10 binding activity, AF10-MLL or other MLL fusion proteinbinding activity, and/or any other biological activity of interest. ADOT1L polypeptide can be a functional fragment of a full-length DOT1Lpolypeptide or functional equivalent thereof, and may comprise any DOT1domain of interest, including but not limited to the catalytic domain,the SAM binding domain and/or the positively charged domain, the AF10interaction domain and/or a nuclear export signal.

Methods of assessing DOT1L binding to histones, nucleosomes, nucleicacids or polypeptides can be carried out using standard techniques thatwill be apparent to those skilled in the art (see the Exemplificationfor exemplary methods). Such methods include yeast and mammaliantwo-hybrid assays and co-immunoprecipitation techniques.

For example, a compound that modulates DOT1L H3K79 HMTase activity canbe verified by: contacting a DOT1L polypeptide with a histone or peptidesubstrate comprising H3 in the presence of a test compound; detectingthe level of H3K79 methylation of the histone or peptide substrate underconditions sufficient to provide H3K79 methylation, wherein an elevationor reduction in H3K79 methylation in the presence of the test compoundas compared with the level of histone H3K79 methylation in the absenceof the test compound indicates that the test compound modulates DOT1LH3K79 HMTase activity.

The screening methods of the invention can be carried out in acell-based or cell-free system. As a further alternative, the assay canbe performed in a whole animal (including transgenic non-human animals).Further, with respect to cell-based systems, the DOT1L polypeptide (orany other polypeptide used in the assay) can be added directly to thecell or can be produced from a nucleic acid in the cell. The nucleicacid can be endogenous to the cell or can be foreign (e.g., agenetically modified cell).

Any compound of interest can be screened according to the presentinvention. Suitable test compounds include small organic compounds.Small organic compounds include a wide variety of organic molecules,such as heterocyclics, aromatics, alicyclics, aliphatics andcombinations thereof, comprising steroids, antibiotics, enzymeinhibitors, ligands, hormones, drugs, alkaloids, opioids, terpenes,porphyrins, toxins, catalysts, as well as combinations thereof.

DEFINITIONS

For convenience, certain terms employed in the specification, examples,and appended claims are collected here. All definitions, as defined andused herein, supersede dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e., “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

The terms “co-administration” and “co-administering” refer to bothconcurrent administration (administration of two or more therapeuticagents at the same time) and time varied administration (administrationof one or more therapeutic agents at a time different from that of theadministration of an additional therapeutic agent or agents), as long asthe therapeutic agents are present in the patient to some extent at thesame time.

The term “hydrate” refers to a pharmaceutically acceptable form of aspecified compound, with one or more water molecules, that retains thebiological effectiveness of such compound.

The definition of each expression, e.g., alkyl, m, n, and the like, whenit occurs more than once in any structure, is intended to be independentof its definition elsewhere in the same structure.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., a compound whichdoes not spontaneously undergo transformation such as by rearrangement,cyclization, elimination, or other reaction.

The term “substituted” is also contemplated to include all permissiblesubstituents of organic compounds. In a broad aspect, the permissiblesubstituents include acyclic and cyclic, branched and unbranched,carbocyclic and heterocyclic, aromatic and nonaromatic substituents oforganic compounds. Illustrative substituents include, for example, thosedescribed herein below. The permissible substituents may be one or moreand the same or different for appropriate organic compounds. Forpurposes of this invention, the heteroatoms such as nitrogen may havehydrogen substituents and/or any permissible substituents of organiccompounds described herein which satisfy the valences of theheteroatoms. This invention is not intended to be limited in any mannerby the permissible substituents of organic compounds.

The term “lower” when appended to any of the groups listed belowindicates that the group contains less than seven carbons (i.e., sixcarbons or less). For example “lower alkyl” refers to an alkyl groupcontaining 1-6 carbons, and “lower alkenyl” refers to an alkenyl groupcontaining 2-6 carbons.

The term “unsaturated,” as used herein, pertains to compounds and/orgroups which have at least one carbon-carbon double bond orcarbon-carbon triple bond.

The term “aliphatic,” as used herein, pertains to compounds and/orgroups which are linear or branched, but not cyclic (also known as“acyclic” or “open-chain” groups).

The term “cyclic,” as used herein, pertains to compounds and/or groupswhich have one ring, or two or more rings (e.g., spiro, fused, bridged).

The term “aromatic” refers to a planar or polycyclic structurecharacterized by a cyclically conjugated molecular moiety containing4n+2 electrons, wherein n is the absolute value of an integer. Aromaticmolecules containing fused, or joined, rings also are referred to asbicyclic aromatic rings. For example, bicyclic aromatic rings containingheteroatoms in a hydrocarbon ring structure are referred to as bicyclicheteroaryl rings.

The term “hydrocarbon” as used herein refers to an organic compoundconsisting entirely of hydrogen and carbon.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

The term “heteroatom” as used herein is art-recognized and refers to anatom of any element other than carbon or hydrogen. Illustrativeheteroatoms include boron, nitrogen, oxygen, phosphorus, sulfur andselenium.

The term “alkyl” means an aliphatic hydrocarbon radical containing from1 to 20 carbon atoms. In one embodiment the term “alkyl” refers to analiphatic hydrocarbon radical containing from 1 to 15 carbon atoms. Inone embodiment the term “alkyl” refers to an aliphatic hydrocarbonradical containing from 1 to 10 carbon atoms. In one embodiment the term“alkyl” refers to an aliphatic hydrocarbon radical containing from 1 to6 carbon atoms. Representative examples of alkyl include, but are notlimited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl,iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl,2-methylcyclopentyl, 1-(1-ethylcyclopropyl)ethyl and 1-cyclohexylethyl.

The term “cycloalkyl” refers to a cyclic hydrocarbon radical containingfrom 3 to 15 carbon atoms. In one embodiment the term “cycloalkyl”refers to a cyclic hydrocarbon radical containing from 3 to 10 carbonatoms. In one embodiment the term “cycloalkyl” refers to a cyclichydrocarbon radical containing from 3 to 7 carbon atoms. Representativeexamples of cycloalkyl include, but are not limited to, cyclopropyl andcyclobutyl.

The term “alkenyl” as used herein means a straight or branched chainhydrocarbon radical containing from 2 to 10 carbons and containing atleast one carbon-carbon double bond formed by the removal of twohydrogens. Representative examples of alkenyl include, but are notlimited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl,4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, and 3-decenyl.

The term “alkynyl” as used herein means a straight or branched chainhydrocarbon radical containing from 2 to 10 carbon atoms and containingat least one carbon-carbon triple bond. Representative examples ofalkynyl include, but are not limited, to acetylenyl, 1-propynyl,2-propynyl, 3-butynyl, 2-pentynyl, and 1-butynyl.

The term “alkylene,” is art-recognized, and as used herein pertains to adiradical obtained by removing two hydrogen atoms of an alkyl group, asdefined above.

The term “carbocyclyl” as used herein means a monocyclic or multicyclic(e.g., bicyclic, tricyclic, etc.) hydrocarbon radical containing from 3to 12 carbon atoms that is completely saturated or has one or moreunsaturated bonds, and for the avoidance of doubt, the degree ofunsaturation does not result in an aromatic ring system (e.g., phenyl).Examples of carbocyclyl groups include 1-cyclopropyl, 1-cyclobutyl,2-cyclopentyl, 1-cyclopentenyl, 3-cyclohexyl, 1-cyclohexenyl and2-cyclopentenylmethyl.

The term “heterocyclyl”, as used herein refers to a radical of anon-aromatic, ring system, including, but not limited to, monocyclic,bicyclic and tricyclic rings, which can be completely saturated or whichcan contain one or more units of unsaturation, for the avoidance ofdoubt, the degree of unsaturation does not result in an aromatic ringsystem, and have 3 to 12 atoms including at least one heteroatom, suchas nitrogen, oxygen, or sulfur. For purposes of exemplification, whichshould not be construed as limiting the scope of this invention, thefollowing are examples of heterocyclic rings: aziridinyl, azirinyl,oxiranyl, thiiranyl, thiirenyl, dioxiranyl, diazirinyl, azetyl,oxetanyl, oxetyl, thietanyl, thietyl, diazetidinyl, dioxetanyl,dioxetenyl, dithietanyl, dithietyl, furyl, dioxalanyl, pyrrolyl,oxazolyl, thiazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, triazolyl,triazinyl, isothiazolyl, isoxazolyl, thiophenyl, pyrazolyl, tetrazolyl,pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, tetrazinyl,quinolinyl, isoquinolinyl, quinoxalinyl, quinazolinyl, pyridopyrazinyl,benzoxazolyl, benzothiophenyl, benzimidazolyl, benzothiazolyl,benzoxadiazolyl, benzthiadiazolyl, indolyl, benztriazolyl,naphthyridinyl, azepines, azetidinyl, morpholinyl, oxopiperidinyl,oxopyrrolidinyl, piperazinyl, piperidinyl, pyrrolidinyl, quinicludinyl,thiomorpholinyl, tetrahydropyranyl and tetrahydropyranyl. Theheterocyclyl groups of the invention are substituted with 0, 1, 2, 3, 4or 5 substituents independently selected from the group consisting ofalkyl, alkenyl, alkynyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy,alkyenyloxy, alkynyloxy, carbocyclyloxy, heterocyclyloxy, haloalkoxy,fluoroalkyloxy, sulfhydryl, alkylthio, haloalkylthio, fiuoroalkylthio,alkyenylthio, alkynylthio, sulfonic acid, alkylsulfonyl,haloalkylsulfonyl, fluoroalkylsulfonyl, alkenylsulfonyl,alkynylsulfonyl, alkoxysulfonyl, haloalkoxysulfonyl,fiuoroalkoxysulfonyl, alkenyloxysulfonyl, alkynyloxysulfony,aminosulfonyl, sulfinic acid, alkylsulfinyl, haloalkylsulfinyl,fluoroalkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, alkoxysulfinyl,haloalkoxysulfinyl, fluoroalkoxysulfinyl, alkenyloxysulfinyl,alkynyloxysulfiny, aminosulfinyl, formyl, alkylcarbonyl,haloalkylcarbonyl, fluoroalkylcarbonyl, alkenylcarbonyl,alkynylcarbonyl, carboxy, alkoxycarbonyl, haloalkoxycarbonyl,fluoroalkoxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl,alkylcarbonyloxy, haloalkylcarbonyloxy, fluoroalkylcarbonyloxy,alkenylcarbonyloxy, alkynylcarbonyloxy, alkylsulfonyloxy,haloalkylsulfonyloxy, fluoroalkylsulfonyloxy, alkenylsulfonyloxy,alkynylsulfonyloxy, haloalkoxysulfonyloxy, fiuoroalkoxysulfonyloxy,alkenyloxysulfonyloxy, alkynyloxysulfonyloxy, alkylsulfinyloxy,haloalkylsulfinyloxy, fiuoroalkylsulfinyloxy, alkenylsulfinyloxy,alkynylsulfinyloxy, alkoxysulfinyloxy, haloalkoxysulfinyloxy,fluoroalkoxysulfinyloxy, alkenyloxysulfinyloxy, alkynyloxysulfinyloxy,aminosulfinyloxy, amino, amido, aminosulfonyl, aminosulfinyl, cyano,nitro, azido, phosphinyl, phosphoryl, silyl, silyloxy, and any of saidsubstiuents bound to the heterocyclyl group through an alkylene moiety(e.g., methylene).

The term “aryl,” as used herein means a phenyl, naphthyl, phenanthrenyl,or anthracenyl group. The aryl groups of the present invention can beoptionally substituted with 1, 2, 3, 4 or 5 substituents independentlyselected from the group consisting of alkyl, alkenyl, alkynyl, halo,haloalkyl, fluoroalkyl, hydroxy, alkoxy, alkyenyloxy, alkynyloxy,carbocyclyloxy, heterocyclyloxy, haloalkoxy, fluoroalkyloxy, sulfhydryl,alkylthio, haloalkylthio, fluoroalkylthio, alkyenylthio, alkynylthio,sulfonic acid, alkylsulfonyl, haloalkylsulfonyl, fluoroalkylsulfonyl,alkenylsulfonyl, alkynylsulfonyl, alkoxysulfonyl, haloalkoxysulfonyl,fluoroalkoxysulfonyl, alkenyloxysulfonyl, alkynyloxysulfony,aminosulfonyl, sulfinic acid, alkylsulfinyl, haloalkylsulfinyl,fluoroalkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, alkoxysulfinyl,haloalkoxysulfinyl, fluoroalkoxysulfinyl, alkenyloxysulfinyl,alkynyloxysulfiny, aminosulfinyl, formyl, alkylcarbonyl,haloalkylcarbonyl, fluoroalkylcarbonyl, alkenylcarbonyl,alkynylcarbonyl, carboxy, alkoxycarbonyl, haloalkoxycarbonyl,fluoroalkoxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl,alkylcarbonyloxy, haloalkylcarbonyloxy, fluoroalkylcarbonyloxy,alkenylcarbonyloxy, alkynylcarbonyloxy, alkylsulfonyloxy,haloalkylsulfonyloxy, fluoroalkylsulfonyloxy, alkenylsulfonyloxy,alkynylsulfonyloxy, haloalkoxysulfonyloxy, fluoroalkoxysulfonyloxy,alkenyloxysulfonyloxy, alkynyloxysulfonyloxy, alkylsulfinyloxy,haloalkylsulfinyloxy, fluoroalkylsulfinyloxy, alkenylsulfinyloxy,alkynylsulfinyloxy, alkoxysulfinyloxy, haloalkoxysulfinyloxy,fluoroalkoxysulfinyloxy, alkenyloxysulfinyloxy, alkynyloxysulfinyloxy,aminosulfinyloxy, amino, amido, aminosulfonyl, aminosulfinyl, cyano,nitro, azido, phosphinyl, phosphoryl, silyl, silyloxy, and any of saidsubstituents bound to the heterocyclyl group through an alkylene moiety(e.g., methylene).

The term “arylene,” is art-recognized, and as used herein pertains to adiradical obtained by removing two hydrogen atoms of an aryl ring, asdefined above.

The term “arylalkyl” or “aralkyl” as used herein means an aryl group, asdefined herein, appended to the parent molecular moiety through an alkylgroup, as defined herein. Representative examples of aralkyl include,but are not limited to, benzyl, 2-phenylethyl, 3-phenylpropyl, and2-naphth-2-ylethyl.

The term “biaryl,” as used herein means an aryl-substituted aryl, anaryl-substituted heteroaryl, a heteroaryl-substituted aryl or aheteroaryl-substituted heteroaryl, wherein aryl and heteroaryl are asdefined herein. Representative examples include 4-(phenyl)phenyl and4-(4-methoxyphenyl)pyridinyl.

The term “fused bicyclyl” as used herein means the radical of a bicyclicring system wherein the two rings are ortho-fused, and each ring,contains a total of four, five, six or seven atoms (i.e. carbons andheteroatoms) including the two fusion atoms, and each ring can becompletely saturated, can contain one or more units of unsaturation, orcan be completely unsaturated (e.g., in some case, aromatic). For theavoidance of doubt, the degree of unsaturation in the fused bicyclyldoes not result in an aryl or heteroaryl moiety.

The term “heteroaryl” as used herein include radicals of aromatic ringsystems, including, but not limited to, monocyclic, bicyclic andtricyclic rings, which have 3 to 12 atoms including at least oneheteroatom, such as nitrogen, oxygen, or sulfur. For purposes ofexemplification, which should not be construed as limiting the scope ofthis invention: aminobenzimidazole, benzimidazole, azaindolyl,benzo[b]thienyl, benzimidazolyl, benzofuranyl, benzoxazolyl,benzothiazolyl, benzothiadiazolyl, benzotriazolyl, benzoxadiazolyl,furanyl, imidazolyl, imidazopyridinyl, indolyl, indolinyl, indazolyl,isoindolinyl, isoxazolyl, isothiazolyl, isoquinolinyl, oxadiazolyl,oxazolyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridinyl,pyrimidinyl, pyrrolyl, pyrrolo[2,3-d]pyrimidinyl,pyrazolo[3,4-d]pyrimidinyl, quinolinyl, quinazolinyl, triazolyl,thiazolyl, thiophenyl, tetrahydroindolyl, tetrazolyl, thiadiazolyl,thienyl, thiomorpholinyl, triazolyl or tropanyl. The heteroaryl groupsof the invention are substituted with 0, 1, 2, 3, 4 or 5 substituentsindependently selected from the group consisting of alkyl, alkenyl,alkynyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, alkyenyloxy,alkynyloxy, carbocyclyloxy, heterocyclyloxy, haloalkoxy, fluoroalkyloxy,sulfhydryl, alkylthio, haloalkylthio, fluoroalkylthio, alkyenylthio,alkynylthio, sulfonic acid, alkylsulfonyl, haloalkylsulfonyl,fluoroalkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, alkoxysulfonyl,haloalkoxysulfonyl, fluoroalkoxysulfonyl, alkenyloxysulfonyl,alkynyloxysulfony, aminosulfonyl, sulfinic acid, alkylsulfinyl,haloalkylsulfinyl, fluoroalkylsulfinyl, alkenylsulfinyl,alkynylsulfinyl, alkoxysulfinyl, haloalkoxysulfinyl,fluoroalkoxysulfinyl, alkenyloxysulfinyl, alkynyloxysulfiny,aminosulfinyl, formyl, alkylcarbonyl, haloalkylcarbonyl,fluoroalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, carboxy,alkoxycarbonyl, haloalkoxycarbonyl, fluoroalkoxycarbonyl,alkenyloxycarbonyl, alkynyloxycarbonyl, alkylcarbonyloxy,haloalkylcarbonyloxy, fluoroalkylcarbonyloxy, alkenylcarbonyloxy,alkynylcarbonyloxy, alkylsulfonyloxy, haloalkylsulfonyloxy,fluoroalkylsulfonyloxy, alkenylsulfonyloxy, alkynylsulfonyloxy,haloalkoxysulfonyloxy, fluoroalkoxysulfonyloxy, alkenyloxysulfonyloxy,alkynyloxysulfonyloxy, alkylsulfinyloxy, haloalkylsulfinyloxy,fluoroalkylsulfinyloxy, alkenylsulfinyloxy, alkynylsulfinyloxy,alkoxysulfinyloxy, haloalkoxysulfinyloxy, fluoroalkoxysulfinyloxy,alkenyloxysulfinyloxy, alkynyloxysulfinyloxy, aminosulfinyloxy, amino,amido, aminosulfonyl, aminosulfinyl, cyano, nitro, azido, phosphinyl,phosphoryl, silyl, silyloxy, and any of said substituents bound to theheteroaryl group through an alkylene moiety (e.g., methylene).

The term “heteroarylene,” is art-recognized, and as used herein pertainsto a diradical obtained by removing two hydrogen atoms of a heteroarylring, as defined above.

The term “heteroarylalkyl” or “heteroaralkyl” as used herein means aheteroaryl, as defined herein, appended to the parent molecular moietythrough an alkyl group, as defined herein. Representative examples ofheteroarylalkyl include, but are not limited to, pyridin-3-ylmethyl and2-(thien-2-yl)ethyl.

The term “halo” or “halogen” means —Cl, —Br, —I or —F.

The term “haloalkyl” means an alkyl group, as defined herein, wherein atleast one hydrogen is replaced with a halogen, as defined herein.Representative examples of haloalkyl include, but are not limited to,chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, and2-chloro-3-fluoropentyl.

The term “fluoroalkyl” means an alkyl group, as defined herein, whereinsome or all of the hydrogens are replaced with fluorines.

The term “haloalkylene,” as used herein pertains to diradical obtainedby removing two hydrogen atoms of an haloalkyl group, as defined above.

The term “hydroxy” as used herein means an —OH group.

The term “alkoxy” as used herein means an alkyl group, as definedherein, appended to the parent molecular moiety through an oxygen atom.Representative examples of alkoxy include, but are not limited to,methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, andhexyloxy. The terms “alkyenyloxy”, “alkynyloxy”, “carbocyclyloxy”, and“heterocyclyloxy” are likewise defined.

The term “haloalkoxy” as used herein means an alkoxy group, as definedherein, wherein at least one hydrogen is replaced with a halogen, asdefined herein. Representative examples of haloalkoxy include, but arenot limited to, chloromethoxy, 2-fluoroethoxy, trifluoromethoxy, andpentafluoroethoxy. The term “fluoroalkyloxy” is likewise defined.

The term “aryloxy” as used herein means an aryl group, as definedherein, appended to the parent molecular moiety through an oxygen. Theterm “heteroaryioxy” as used herein means a heteroaryl group, as definedherein, appended to the parent molecular moiety through an oxygen. Theterms “heteroaryloxy” is likewise defined.

The term “arylalkoxy” or “arylalkyloxy” as used herein means anarylalkyl group, as defined herein, appended to the parent molecularmoiety through an oxygen. The term “heteroarylalkoxy” is likewisedefined. Representative examples of aryloxy and heteroarylalkoxyinclude, but are not limited to, 2-chlorophenylmethoxy,3-trifluoromethyl-phenylethoxy, and 2,3-dimethylpyridinylmethoxy.

The term “sulfhydryl” or “thio” as used herein means a —SH group.

The term “alkylthio” as used herein means an alkyl group, as definedherein, appended to the parent molecular moiety through a sulfur.Representative examples of alkylthio include, but are not limited,methylthio, ethylthio, tert-butylthio, and hexylthio. The terms“haloalkylthio”, “fluoroalkylthio”, “alkyenylthio”, “alkynylthio”,“carbocyclylthio”, and “heterocyclylthio” are likewise defined.

The term “arylthio” as used herein means an aryl group, as definedherein, appended to the parent molecular moiety through an sulfur. Theterm “heteroarylthio” is likewise defined.

The term “arylalkylthio” or “aralkylthio” as used herein means anarylalkyl group, as defined herein, appended to the parent molecularmoiety through an sulfur. The term “heteroarylalkylthio” is likewisedefined.

The term “sulfonyl” as used herein refers to —S(═O)₂— group.

The term “sulfonic acid” as used herein refers to —S(═O)₂OH.

The term “alkylsulfonyl” as used herein means an alkyl group, as definedherein, appended to the parent molecular moiety through a sulfonylgroup, as defined herein. Representative examples of alkylsulfonylinclude, but are not limited to, methylsulfonyl and ethylsulfonyl. Theterms “haloalkylsulfonyl”, “fluoroalkylsulfonyl”, “alkenylsulfonyl”,“alkynylsulfonyl”, “carbocyclylsulfonyl”, “heterocyclylsulfonyl”,“arylsulfonyl”, “aralkylsulfonyl”, “heteroarylsulfonyl” and“heteroaralkylsulfonyl” are likewise defined.

The term “alkoxysulfonyl” as used herein means an alkoxy group, asdefined herein, appended to the parent molecular moiety through asulfonyl group, as defined herein. Representative examples ofalkoxysulfonyl include, but are not limited to, methoxysulfonyl,ethoxysulfonyl and propoxysulfonyl. The terms “haloalkoxysulfonyl”,“fluoroalkoxysulfonyl”, “alkenyloxysulfonyl”, “alkynyloxysulfonyl”,“carbocyclyloxysulfonyl”, “heterocyclyloxysulfonyl”, “aryloxysulfonyl”,“aralkyloxysulfonyl”, “heteroaryloxysulfonyl” and“heteroaralkyloxysulfonyl” are likewise defined.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized andrefer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl,and nonafluorobutanesulfonyl groups, respectively. The terms triflate,tosylate, mesylate, and nonaflate are art-recognized and refer totrifluoromethanesulfonate ester, p-toluenesulfonate ester,methanesulfonate ester, and nonafluorobutanesulfonate ester functionalgroups and molecules that contain said groups, respectively.

The term “aminosulfonyl” as used herein means an amino group, as definedherein, appended to the parent molecular moiety through a sulfonylgroup.

The term “sulfonyl” as used herein refers to —S(═O)— group. Sulfinylgroups are as defined above for sulfonyl groups. The term “sulfinicacid” as used herein refers to —S(═O)OH.

The term “oxy” refers to a —O— group.

The term “carbonyl” as used herein means a —C(═O)— group.

The term “thiocarbonyl” as used herein means a —C(═S)— group.

The term “formyl” as used herein means a —C(═O)H group.

The term “alkylcarbonyl” as used herein means an alkyl group, as definedherein, appended to the parent molecular moiety through a carbonylgroup, as defined herein. Representative examples of alkylcarbonylinclude, but are not limited to, acetyl, 1-oxopropyl,2,2-dimethyl-1-oxopropyl, 1-oxobutyl, and 1-oxopentyl. The terms“haloalkylcarbonyl”, “fluoroalkylcarbonyl”, “alkenyl carbonyl”,“alkynylcarbonyl”, “carbocyclylcarbonyl”, “heterocyclylcarbonyl”,“arylcarbonyl”, “aralkylcarbonyl”, “heteroarylcarbonyl”, and“heteroaralkylcarbonyl” are likewise defined.

The term “carboxy” as used herein means a —CO₂H group.

The term “alkoxycarbonyl” as used herein means an alkoxy group, asdefined herein, appended to the parent molecular moiety through acarbonyl group, as defined herein. Representative examples ofalkoxycarbonyl include, but are not limited to, methoxycarbonyl,ethoxycarbonyl, and tert-butoxycarbonyl. The terms “haloalkoxycarbonyl”,“fluoroalkoxycarbonyl”, “alkenyloxycarbonyl”, “alkynyloxycarbonyl”,“carbocyclyloxycarbonyl”, “heterocyclyloxycarbonyl”, “aryloxycarbonyl”,“aralkyloxycarbonyl”, “heteroaryloxycarbonyl”, and“heteroaralkyloxycarbonyl” are likewise defined.

The term “alkylcarbonyloxy” as used herein means an alkylcarbonyl group,as defined herein, appended to the parent molecular moiety through anoxygen atom. Representative examples of alkylcarbonyloxy include, butare not limited to, acetyloxy, ethylcarbonyloxy, andtert-butylcarbonyloxy. The terms “haloalkylcarbonyloxy”,“fluoroalkylcarbonyloxy”, “alkenylcarbonyloxy”, “alkynylcarbonyloxy”,“carbocyclylcarbonyloxy”, “heterocyclylcarbonyloxy”, “arylcarbonyloxy”,“aralkylcarbonyloxy”, “heteroarylcarbonyloxy”, and“heteroaralkylcarbonyloxy” are likewise defined.

The term “alkylsulfonyloxy” as used herein means an alkylsulfonyl group,as defined herein, appended to the parent molecular moiety through anoxygen atom. The terms “haloalkylsulfonyloxy”, “fluoroalkylsulfonyloxy”,“alkenylsulfonyloxy”, “alkynylsulfonyloxy”, “carbocyclylsulfonyloxy”,“heterocyclylsulfonyloxy”, “arylsulfonyloxy”, “aralkylsulfonyloxy”,“heteroarylsulfonyloxy”, “heteroaralkylsulfonyloxy”,“haloalkoxysulfonyloxy”, “fluoroalkoxysulfonyloxy”,“alkenyloxysulfonyloxy”, “alkynyloxysulfonyloxy”,“carbocyclyloxysulfonyloxy”, “heterocyclyloxysulfonyloxy”,“aryloxysulfonyloxy”, “aralkyloxysulfonyloxy”,“heteroaryloxysulfonyloxy” and “heteroaralkyloxysulfonyloxy” arelikewise defined.

The term “amino” as used herein refers to —NH₂ and substitutedderivatives thereof wherein one or both of the hydrogens areindependently replaced with substituents selected from the groupconsisting of alkyl, haloalkyl, fluoroalkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl,alkylcarbonyl, haloalkylcarbonyl, fluoroalkylcarbonyl, alkenylcarbonyl,alkynylcarbonyl, carbocyclylcarbonyl, heterocyclylcarbonyl,arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyland the sulfonyl and sulfinyl groups defined above; or when bothhydrogens together are replaced with an alkylene group (to form a ringwhich contains the nitrogen). Representative examples include, but arenot limited to methylamino, acetylamino, and dimethylamino.

The term “amido” as used herein means an amino group, as defined herein,appended to the parent molecular moiety through a carbonyl.

The term “cyano” as used herein means a —C≡N group.

The term “nitro” as used herein means a —NO₂ group.

The term “azido” as used herein means a —N₃ group.

The term “phosphinyl” or “phosphine” as used herein includes —PH₃ andsubstituted derivatives thereof wherein one, two or three of thehydrogens are independently replaced with substituents selected from thegroup consisting of alkyl, haloalkyl, fluoroalkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl,alkoxy, haloalkoxy, fluoroalkyloxy, alkenyloxy, alkynyloxy,carbocyclyloxy, heterocyclyloxy, aryloxy, aralkyloxy, heteroaryloxy,heteroaralkyloxy, and amino.

The term “phosphoryl” as used herein refers to —P(═O)OH₂ and substitutedderivatives thereof wherein one or both of the hydroxyls areindependently replaced with substituents selected from the groupconsisting of alkyl, haloalkyl, fluoroalkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl,alkoxy, haloalkoxy, fluoroalkyloxy, alkenyloxy, alkynyloxy,carbocyclyloxy, heterocyclyloxy, aryloxy, aralkyloxy, heteroaryloxy,heteroaralkyloxy, and amino.

The term “silyl” as used herein includes H₃Si— and substitutedderivatives thereof wherein one, two or three of the hydrogens areindependently replaced with substituents selected from alkyl, haloalkyl,fluoroalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, aralkyl,heteroaryl, and heteroaralkyl. Representative examples includetrimethylsilyl (TMS), tert-butyldiphenylsilyl (TBDPS),tert-butyldimethylsilyl (TBS/TBDMS), triisopropylsilyl (TIPS), and[2-(trimethylsilyl)ethoxy]methyl (SEM).

The term “silyloxy” as used herein means a silyl group, as definedherein, is appended to the parent molecule through an oxygen atom.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms represent methyl,ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations.

The term “treating” as used herein, encompasses the administrationand/or application of one or more compounds described herein, to asubject, for the purpose of providing prevention of or management of,and/or remedy for a condition. “Treatment” for the purposes of thisdisclosure, may, but does not have to, provide a cure; rather,“treatment” may be in the form of management of the condition. When thecompounds described herein are used to treat unwanted proliferatingcells, including cancers, “treatment” includes partial or totaldestruction of the undesirable proliferating cells with minimaldestructive effects on normal cells. A desired mechanism of treatment ofunwanted rapidly proliferating cells, including cancer cells, at thecellular level is apoptosis.

The term “preventing” as used herein includes either preventing orslowing the onset of a clinically evident disease progression altogetheror preventing or slowing the onset of a preclinically evident stage of adisease in individuals at risk. This includes prophylactic treatment ofthose at risk of developing a disease.

The term “subject” for purposes of treatment includes any human oranimal subject who has been diagnosed with, has symptoms of or is atrisk of developing a disorder. For methods of prevention the subject isany human or animal subject. To illustrate, for purposes of prevention,a subject may be a human subject who is at risk of or is geneticallypredisposed to obtaining a disorder characterized by unwanted, rapidcell proliferation, such as cancer. The subject may be at risk due toexposure to carcinogenic agents, being genetically predisposed todisorders characterized by unwanted, rapid cell proliferation, and soon. Besides being useful for human treatment, the compounds describedherein are also useful for veterinary treatment of mammals, includingcompanion animals and farm animals, such as, but not limited to dogs,cats, horses, cows, sheep, and pigs.

Except as otherwise indicated, standard methods can be used for theproduction of recombinant and synthetic polypeptides, fusion proteins,antibodies or antigen-binding fragments thereof, manipulation of nucleicacid sequences, production of transformed cells, and the like. Suchtechniques are known to those skilled in the art. See, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual 2nd Ed. (Cold Spring Harbor,N.Y., 1989); F. M. Ausubel et al., Current Protocols in MolecularBiology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc.,New York).

The term “DOT1L polypeptide” encompasses functional fragments of thefull-length polypeptides and functional equivalents of either of theforegoing that have substantially similar or substantially identicalamino acid sequences (at least about 75%, 80%, 85%, 90%, 95% 98% or moreamino acid sequence similarity or identity), where the functionalfragment or functional equivalent retains one or more of the functionalproperties of the native polypeptide.

By “functional” it is meant that the polypeptide (or nucleic acid) hasthe same or substantially similar activity with respect to one or moreof the biological properties of the native polypeptide (or nucleicacid), e.g., at least about 50%, 75%, 85%, 90%, 95% or 98% or more ofthe activity of the native polypeptide (or nucleic acid).

The term “modulate” (and grammatical equivalents) refers to an increaseor decrease in activity. In particular embodiments, the term “increase”or “enhance” (and grammatical equivalents) means an elevation by atleast about 25%, 50%, 75%, 2-fold, 3-fold, 5-fold, 10-fold, 15-fold,20-fold or more. In particular embodiments, the terms “decrease” or“reduce” (and grammatical equivalents) means a diminishment by at leastabout 25%, 40%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, 98% or more. In someembodiments, the indicated activity, substance or other parameter is notdetectable. Specifically provided are inhibitors of DOT1L.

The term “pharmacodynamic marker” refers to a molecular marker of drugresponse that can be measured in patients receiving the drug. The markershould be a direct measure of modulation of the drug target and be ableto show quantitative changes in response to dose. A potentialpharmacodynamic marker for a DOT1L inhibitor could be levels of histoneH3K79 methylation in disease or surrogate tissue.

Exemplification

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

ABBREVIATIONS Abbreviation Definition AA ammonium acetate Ac acetyl ACNacetonitrile AcOH acetic acid atm atmosphere Bn benzyl BOC tert-butoxycarbonyl BOP (benzotriazol-1-yloxy)tris(dimethylamino)phos- phoniumhexafluorophosphate Cbz benzyloxy carbonyl COMU(1-Cyano-2-ethoxy-2-oxoethylidenamino-oxy)dimethylamino-morpholino-carbenium hexafluorophosphate d days DBU1,8-diazabicyclo[5.4.0]undec-7-ene DCE 1,2 dichloroethane DCMdichloromethane DEAD Diethyl azodicarboxylate DIAD Diisopropylazodicarboxylate DiBAL-H di-isobutyl aluminum hydride DIPEAN,N-diisopropylethylamine (Hunig's base) DMAPN,N-dimethyl-4-aminopyridine DMB 2,4 dimethoxy benzyl DMFdimethylformamide DMSO Dimethyl sulfoxide DPPA Diphenylphosphonic azideEA or EtOAc Ethyl acetate EDC or EDCIN-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide ELS Evaporative LightScattering ESI− Electrospray negative mode ESI+ Electrospray positivemode Et₂O diethyl ether Et₃N or TEA triethylamine EtOH ethanol FA formicacid FC Flash chromatography h hours H₂O water HATUO-(7-Azabenzotriazol-1-yl)-N,N,N′,N′- tetramethyluroniumhexafluorophosphate HCl hydrochloric acid HOAT1-Hydroxy-7-azabenzotriazole HOBt 1-Hydroxybenzotriazole HO-SuN-Hydroxysuccinimide HPLC High performance liquid chromatography KHMDsPotassium hexamethyldisilazide LC/MS or LC-MS liquid chromatography massspectrum LDA Lithium diisopropylamide LG leaving group LiHMDs Lithiumhexamethyldisilazide M Molar m/z mass/charge ratio m-CPBAmeta-chloroperbenzoic acid MeCN Acetonitrile MeOD d₄-methanol MeOHmethanol MgSO₄ magnesium sulfate min minutes MS Mass Spectrometry MsMesyl MS mass spectrum MsCl Mesyl chloride MsO Mesylate MWI microwaveirradiation Na₂CO₃ sodium carbonate NaHCO₃ sodium bicarbonate NaHMDsSodium hexamethyldisilazide NaOH sodium hydroxide NIS N-iodosuccinimideNMR Nuclear Magnetic Resonance o/n or O/N overnight PE Petroleum EtherPG protecting group PMB para methoxybenzyl PPAA 1-Propanephosphonic acidcyclic anhydride ppm parts per million prep HPLC preparative Highperformance liquid chromatography prep TLC preparative thin layerchromatography p-TsOH para-toluenesulfonic acid rt or RT roomtemperature SEM 2-(Trimethylsilyl)ethoxymethyl SEMCl-(Trimethylsilyl)ethoxymethyl chloride SFC Super critical chromatographySGC silica gel chromatography STAB Sodium triacetoxy borohydride TBAFtetra-n-butylammonium fluoride TFA trifluoroacetic acid TfO triflate THFtetrahydrofuran THP tetrahydropyran TLC thin layer chromatography Tstosyl TsOH tosic acid UV ultravioletGeneral Methods

Cell Culture.

Human Leukemia cell lines THP-1, RS4; 11, and MV4-11 were obtained fromATCC, MOLM-13 cells were obtained from DSMZ. All lines were grown inRPMI 1640 containing 10% FBS and maintained using the vendorsrecommended cell densities and environmental conditions. Media wassupplemented with non essential amino acids and L-Glutamine. THP-1 cellswere also supplemented with 0.05 mM β-Mercaptoethanol.

Methylation Analysis.

Cells were seeded at 5×10⁵ cells/mL in a 12 well plate at a final volumeof 2 mLs. Cells were dosed with compounds to the appropriateconcentration from a 50 mM DMSO stock solution. Compound and media wererefreshed every two days over the course of seven day incubation bycounting cells using trypan blue exclusion (Vicell), pelleting at 200 gfor 5 minutes and resuspending in fresh media containing compound at afinal cell concentration of 5×10⁵ cells/mL. Following compoundincubation, histones were extracted from 1×10⁶ cells using a commercialhistone extraction kit (Active Motif). Purified histones werequantitated using the BCA protein assay (Pierce) with a BSA standardcurve. 400 ng of isolated histones were fractionated by SDS-PAGE on a4-20% gel and transferred to nitrocellulose membranes. Membranes wereincubated with various primary and secondary antibodies and imaged onthe Licor imaging system (Odyssey). The H3K79-Me2 rabbit polyclonal waspurchased from Abeam. Other rabbit polyclonal antibodies includingH3K4-Me3, H3K9-Me3, H3K27-Me2, and H3K27-Me3 were purchased from CellSignaling Technologies (CST). A mouse monoclonal total H3 antibody wasused as a loading control (CST). Fluorescently labeled secondaryantibodies were purchased from Odyssey.

Cell Growth and Viability Analysis.

Cells were harvested from exponentially growing cell cultures and seededat 3×10⁴ cells per well. Samples were maintained in a 96 well blackwalled clear bottom plate (Corning). A final concentration of 50 uMcompound in 0.2% DMSO was added to the appropriate wells on Day 0.Treatment of MV4-11 and MOLM-13 lasted 14 days, while THP-1 cells weretreated for 18 days. Compound and media were replaced every two daysduring incubation by transferring samples to a V-bottom plate (Corning),spinning at 200 g for 5 minutes in a room temperature rotor,resuspending in fresh media containing compound and transferring back tothe assay plate. Cells were counted periodically using the GuavaViacount assay and read on the EasyCyte Plus instrument (Millipore).Assay plates were split when necessary to within recommended celldensities. Final cell counts were adjusted to take cell splits intoaccount and reported as total viable cells/well

HOXA9 (qPCR).

Cells were treated with compound for 7 days similar to methylationassay. Cell were pelleted at 200 g in a room temperature rotor and totalRNA isolated using the Qiagen RNeasy kit. RNA concentration and qualitywas determined by using the Nanovue (GE Healthcare). Total RNA wasreverse transcribed using a high capacity cDNA reverse transcription kit(Applied Biosystems). A predesigned labeled primer set for HOXA9 waspurchased from Applied Biosystems. qPCR reactions contained 50 ng cDNA,1× labeled primer and 1× Taqman universal PCR master mix (AppliedBiosystems). Samples were run on a 7900 HT Fast Real Time PCR machine(Applied Biosystems) with PCR conditions of 2 min 50° C., 10 min 95° C.,40 cycles at 15 sec 95° C. and 1 min 60° C. HOXA9 cycle numbers werenormalized to the house keeping gene B2 microglobulin (B2M predesignedcontrol from Applied Biosystems). Percent of DMSO control was calculatedwith the equation, percent control=(2^^(−ΔΔCT))*100 where the ΔΔCT isthe difference between normalized HOXA9 sample and control (ΔCTsample−ΔCT control=ΔΔCT).

Determination of IC₅₀.

Compound was serially diluted 3 fold in DMSO for 10 points and 1 μl wasplated in a 384 well microtiter plate. Positive control (100% inhibitionstandard) was 2.5 uM final concentration of S-adenosyl-L-homocysteineand negative control (0% inhibition standard) contained 1 μl of DMSO.Compound was then incubated for 30 minutes with 40 μl per well ofDOT1L(1-416) (0.25 nM final concentration in assay buffer: 20 mM TRIS,pH 8.0, 10 mM NaCl, 0.002% Tween20, 0.005% Bovine Skin Gelatin, 100 mMKCl, and 0.5 mM DTT). 10 μl per well of substrate mix (same assay bufferwith 200 nM S-[methyl-³H]-adenosyl-L methionine, 600 nM of unlabeledS-[methyl-³H]-adenosyl-L methionine, and 20 nM oligonucleosome) wasadded to initiate the reaction. Reaction was incubated for 120 minutesat room temperature and quenched with 10 μl per well of 100 μMS-methyl-adenosyl-L methionine. For detection, substrate from 50 μl ofreaction was immobilized on a 384 well Streptavidin coated Flashplate(Perkin Elmer) (also coated with 0.2% polyethyleneimine) and read on aTop Count scintillation counter (Perkin Elmer).

General Synthetic Schemes

The compounds of the present invention can be prepared in a number ofways known to one skilled in the art of organic synthesis. The compoundsof the present invention can be synthesized using the methods describedbelow, together with synthetic methods known in the art of syntheticorganic chemistry, or by variations thereon as appreciated by thoseskilled in the art. Preferred methods include, but are not limited to,those described below. The reactions are performed in a solventappropriate to the reagents and materials employed and suitable for thetransformations being effected. It will be understood by those skilledin the art of organic synthesis that the functionality present on themolecule should be consistent with the transformations proposed. Thiswill sometimes require a judgment to modify the order of the syntheticsteps or to select one particular process scheme over another in orderto obtain a desired compound of the invention. It will also berecognized that another major consideration in the planning of anysynthetic route in this field is the judicious choice of the protectinggroup used for protection of the reactive functional groups present inthe compounds described in this invention. An authoritative accountdescribing the many alternatives to the trained practitioner is Greeneand Wuts (Protective Groups In Organic Synthesis, Wiley and Sons, 1991).

In the synthetic schemes described herein, compounds may be drawn withone particular configuration for simplicity. Such particularconfigurations are not to be construed as limiting the invention to oneor another isomer, tautomer, regioisomer or stereoisomer, nor does itexclude mixtures of isomers, tautomers, regioisomers or stereoisomers.

Scheme 1 shows the synthesis of modified deazapurine analogs following ageneral route that utilizes well-established chemistry. Condensation ofand tetrahydropyran-2-one with an appropriately substituteddiaminobenzene derivative would provide the benzimidazole (step a).Oxidation with a suitable reagent like IBX in ethyl acetate would givethe modified benzimidazole (step b). Reductive amination with the amineusing sodium acetoxyborohydride in dichloroethane would give coupledproduct (step c). Removal of the acetonide protecting group under acidicconditions using HCl in MeOH would give the desired diol (step d).

Scheme 2 details a synthesis of related deazapurine analogs containingan aminobenzamidazole moiety. Condensation of an amine with4-(1,3-dioxoisoindolin-2-yl)butanal using sodium acetoxyborohydride indichloroethane would give the protected amine (step a). Removal of theamine protecting group would be accomplished by treating thisintermediate with hydrazine in refluxing ethanol and would give the freeamine (step b). Condensation of the amine with an appropriatelysubstituted 2-chlorobenzamidazole at elevated temperature intert-butanol would give the desired aminobenzimidazole (step c). Removalof the acetonide protecting group under acidic conditions using HCl inMeOH would give the desired diol (step d).

Scheme 3 details a synthesis of related deazapurine analogs containingan amino-benzimidazole moiety with a sulfur containing linker. Thestarting thiol would be modified with an appropriate halo ester using amild base like K₂CO₃ in a polar solvent like acetone to give thethioester that would be then saponified with a strong base like LiOH ina polar solvent like MeOH to give the desired acid (Step a). The acidwould be coupled with an appropriate diamine using standard amidecoupling conditions to give the desired amino amide (Step b). The aminoamide would be cyclized to the benzimidazole using a mild acid likeacetic acid as a reagent and solvent to give the benzimidazole (Step c).The oxidation state of the sulfur atom would be adjusted (n=0-2) with avariety of selective oxidation reagents like m-CPBA followed by removalof the acetonide protecting group by treatment with a strong acid likeHCl in a polar solvent like MeOH to give the final product (Step d).

Scheme 4 details a synthesis of related deazapurine analogs containingan aminobenzimidazole moiety with a substituted amine containing linker.The benzyl protected amine would be alkylated with an appropriate haloester in the presence of a mild base like K₂CO₃ in a polar solvent likeacetone to give the desired ester that would be subjected to catalytichydrogenation using hydrogen gas and an appropriate catalyst likepalladium on carbon in a polar solvent like EtOH to give the free amine(Step a). A variety of substituents (R₁) would be introduced usingeither reductive amination conditions or alkylation conditions to givethe R₁ substituted amine. The ester would be then hydrolyzed with astrong base like LiOH in a polar solvent like MeOH to give the acid(Step b). The acid would be coupled with an appropriate diamine usingstandard amide coupling conditions to give the desired amino amide (Stepc). The amino amide would be cyclized to the benzimidazole using a mildacid like acetic acid as a reagent and solvent to give the benzimidazoleand the acetonide protecting group would be removed using a strong acidlike HCl in a polar solvent like MeOH to give the final product (Stepd).

Scheme 5 details a synthesis of related deazapurine analogs containingan aminobenzimidazole moiety with a substituted amide containing linker.Starting with the amine that was previously described in Scheme 2 andtreating with an appropriately substituted acid ester under standardamide coupling conditions would give the amide ester that would behydrolyzed using a strong base like LiOH in a polar solvent like MeOH togive the acid (step a). The acid would be coupled with an appropriatediamine using standard amide coupling conditions to give the desiredamino amide (step b). The amino amide would be cyclized to thebenzimidazole using a mild acid like acetic acid as a reagent andsolvent to give the benzimidazole and the acetonide protecting groupwould be removed using a strong acid like HCl in a polar solvent likeMeOH to give the final product (Step c).

Preparation of Compounds 8 and 9

The following steps are graphically depicted in FIG. 3.

Step 1:(2R,3R,4S,5R)-2-(4-((2,4-dimethoxybenzyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol

A suspension of 7-chloro tubercidin (1.67 g, 5.84 mmol) in 1-butanol(16.0 mL) was treated with N,N-diisopropylethylamine (1.22 mL, 7.01mmol) and 1-(2,4-dimethoxyphenyl)methanamine (1.05 mL, 7.01 mmol) andheated at 100-110° C. overnight. After 20 h, LCMS indicated a newproduct had formed and the starting material was consumed. The mixturewas cooled to room temperature and the solvent removed under highvacuum. The material was purified by flash chromatography (200 g silicagel; 5-10% MeOH/CH₂Cl₂) to yield the title compound (2.19 g, 90%) as afoam: MS (ESI+) for C₂₀H₂₄N₄O₆ m/z 417.1 (M+H)⁺.; (ESI−) for C₂₀H₂₄N₄O₆m/z 415.2 (M−H)⁻; HPLC purity 97% (ret. time, 2.41 min).

Step 2:((3aR,4R,6R,6aR)-6-(4-((2,4-dimethoxybenzyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methanol

A solution of(2R,3R,4S,5R)-2-(4-((2,4-dimethoxybenzyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol(3.30 g, 7.45 mmol) in acetone (76.5 mL) and 2,2-dimethoxypropane (16.5mL, 134 mmol) was treated with 10-camphorsulfonic acid (1.73 g, 7.44mmol) in one portion and the reaction was allowed to stir at roomtemperature. After 1 h, all SM was consumed by HPLC. The reaction wasquenched by the addition of sodium bicarbonate (1.88 g, 22.3 mmol) andthe reaction mixture was stirred for 30 minutes during which time aprecipitate formed. The reaction mixture was partitioned between 200 mLCHCl₃ and 75 mL H₂O. The mixture was diluted with 15 mL brine, extractedand the phases separated. The aqueous phase was washed twice with 50 mLportions of CHCl₃ and the combined organic phase was dried over Na₂SO₄.The solution was filtered and concentrated to yield a foam. The crudeproduct was taken up in methanol (130 mL, 3200 mmol) and treated withp-toluenesulfonic acid monohydrate (1.27 g, 6.70 mmol) in one portion.The mixture was stirred at room temperature for 2 h upon which time thereaction mixture was quenched with sodium bicarbonate (1.88 g, 22.3mmol) and the mixture was stirred for 30 minutes. The solvent wasremoved in vacuo and the residue partitioned between 50 mL H₂O and 150mL CH₂Cl₂ and extracted. The organic phase was washed with 50 mL satNaHCO₃, dried over Na₂SO₄, filtered and concentrated to yield a foam.The product was isolated by flash chromatography (120 g silica gel,60-80% EA/hept) to yield the title compound (2.83 g, 83%) as a lightyellow stiff foam: MS (ESI+) for C₂₃H₂₈N₄O₆ m/z 457.4 (M+H)⁺; (ESI−) forC₂₃H₂₈N₄O₆ m/z 455.2 (M−H)⁻; HPLC purity 99% (ret. time, 3.08 min).

Step 3:N-(((3aR,4R,6R,6aR)-6-(4-((2,4-dimethoxybenzyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-N-methyl-2-nitrobenzenesulfonamide

A solution of((3aR,4R,6R,6aR)-6-(4-((2,4-dimethoxybenzyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methanol(1.52 g, 3.33 mmol) and triphenylphosphine (1.92 g, 7.32 mmol) intetrahydrofuran (25 mL) was cooled at 0° C. and treated dropwise withdiethyl azodicarboxylate (1.26 mL, 7.99 mmol). The now yellow solutionwas treated dropwise with a solution ofN-methyl-2-nitrobenzenesulfonamide (1.01 g, 4.66 mmol) intetrahydrofuran (9.9 mL, 120 mmol) over ˜5 minutes and the solution wasallowed to stir and slowly warm up to room temperature. After 24 h atroom temperature, HPLC indicated that the starting material had beenconsumed. The reaction mixture was partially concentrated and thesolution was purified by flash chromatography (175 g silica gel, 60-90%EA/hept) to yield the title compound (0.82 g, 38%) as a light yellowglass: MS (ESI+) for C₃₀H₃₄N₆O₉S m/z 655.3 (M+H)⁺; (ESI−) forC₃₀H₃₄N₆O₉S m/z 653.3 (M−H)⁻; HPLC purity 68% (ret. time, 3.94 min).

Step 4:N-(2,4-dimethoxybenzyl)-7-((3aR,4R,6R,6aR)-2,2-dimethyl-6-((methylamino)methyl)tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

A suspension ofN-(((3aR,4R,6R,6aR)-6-(4-((2,4-dimethoxybenzyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-N-methyl-2-nitrobenzenesulfonamide(0.82 g, 1.2 mmol) and cesium carbonate (0.82 g, 2.5 mmol) inacetonitrile (23 mL, 440 mmol) was degassed by sparging with nitrogengas for 10 minutes. The solution was treated dropwise with benzenethiol(0.26 mL, 2.5 mmol) and the mixture was allowed to stir at roomtemperature overnight. After 22 h, LCMS indicated the reaction wascomplete. The reaction mixture was partitioned between 60 mL 1N NaOH and120 mL CH₂Cl₂, the layers separated and the aqueous phase was washedwith three 25 mL portions of CH₂Cl₂. The combined organic phase wasdried over MgSO₄, filtered, and concentrated to an oil. The material waspurified by flash chromatography (80 g silica gel, 0-5% 7N NH₃ inCH₃OH/CH₂Cl₂) to yield the title compound (320 mg, 54%) as a foam: MS(ESI+) for C₂₄H₃₁N₅O₆ m/z 470.1 (M+H)⁺; (ESI−) for C₂₄H₃₁N₅O₆ m/z 468.0(M−H)⁻; HPLC purity 99% (ret. time, 2.67 min).

Step 5: 1-(4-tert-butylphenyl)-3-(3-hydroxypropyl)urea

A solution of 3-amino-1-propanol (0.180 mL, 2.36 mmol) in diethylether(15 mL) was cooled at 0° C. and treated dropwise with1-tert-butyl-4-isocyanatobenzene (0.400 mL, 2.25 mmol). The reactionmixture was allowed to slowly warm to room temperature. After 16 h thereaction was found to be complete by TLC (100% ethyl acetate) Thereaction mixture was diluted with 10 mL portions of Cl₂Cl₂ and Et₂O andwashed with 15 mL portions of H₂O, 0.5N HCl and brine and dried overNa₂SO₄. The organic phase was filtered and concentrated to yield acolorless viscous oil which was dissolved in PhCH₃, concentrated andplaced under high vac overnight to yield the title compound (600 mg,110%) as a colorless viscous oil: MS (ESI+) for C₁₄H₂₂N₂O₂ m/z 251.0(M+H)⁺; (ESI−) for O₁₄H₂₂N₂O₂ m/z 249.3 (M−H)⁻; HPLC purity 98% (ret.time, 3.43 min).

Step 6: 3-(3-(4-(tert-butyl)phenyl)ureido)propyl methanesulfonate

A solution of 1-(4-tert-butylphenyl)-3-(3-hydroxypropyl)urea (563 mg,2.25 mmol) in methylene chloride (14 mL) was cooled at 0° C. and treateddropwise with triethylamine (0.376 mL, 2.70 mmol) followed bymethanesulfonyl chloride (0.191 mL, 2.47 mmol) and the mixture wasstirred at 0° C. until complete by TLC. After 30 minutes, the reactionwas complete by TLC (100% EA). The reaction mixture was diluted with 15mL CH₂Cl₂ and the organic phase was washed with 15 mL portions of 1NHCl, sat NaHCO₃ and H₂O and dried over MgSO₄. The solution was filteredand concentrated to a viscous oil that was placed under high vacuum toyield the title compound (800 mg) as a colorless viscous oil that wasstored in the freezer: MS (ESI+) for C₁₅H₂₄N₂O₄S m/z 329.1 (M+H)⁺; HPLCpurity 93% (ret. time, 3.95 min).

Step 7:1-(4-(tert-butyl)phenyl)-3-(3-((((3aR,4R,6R,6aR)-6-(4-((2,4-dimethoxybenzyl)amino)-71′-pyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)(methyl)amino)propyl)urea

A mixture of 3-(3-(4-(tert-butyl)phenyl)ureido)propyl methanesulfonate(224 mg, 0.682 mmol), tetra-n-butylammonium iodide (252 mg, 0.682 mmol)and N,N-diisopropylethylamine (120 μL, 0.67 mmol) was treated withN-(2,4-dimethoxybenzyl)-7-((3aR,4R,6R,6aR)-2,2-dimethyl-6-((methylamino)methyl)tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine(320 mg, 0.68 mmol) in acetonitrile (7.8 mL, 150 mmol) and the solutionwas heated at 65° C. After 16.5 h at 65° C., HPLC indicated the reactionwas about 78-85% complete. An additional 60 mg of mesylate (in 0.4 mLCH₃CN) and 67 mg of TBAI were added and continued heating for 7 h. Thereaction mixture was cooled to room temperature and concentrated. Thecrude residue was purified by flash chromatography (60 g silica gel,3-7% 7N NH₃ in CH₃OH/CH₂Cl₂) to yield the product contaminated withTBAI. The material was dissolved in 20 mL 1/1 ethyl acetate/ethyl etherand washed with three 10 mL portions of H₂O. The organic phase was driedover Na₂SO₄, filtered and concentrated to yield the title compound (295mg, 62%) as a foam: MS (ESI+) for C₃₈H₅₁N₇O₆ m/z 702.2 (M+H)⁺: (ESI−)for C₃₈H₅₁N₇O₆ m/z 700.3 (M−H)⁻; HPLC purity 96% (ret. time, 3.69 min).

Step 8:1-(4-(tert-butyl)phenyl)-3-(3-((((2R,3S,4R,5R)-5-(4-((2,4-dimethoxybenzyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)(methyl)amino)propyl)urea

1-(4-(tert-butyl)phenyl)-3-(3-((((3aR,4R,6R,6aR)-6-(4-((2,4-dimethoxybenzyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)(methyl)amino)propyl)urea(295 mg, 0.420 mmol) was dissolved in trifluoroacetic acid (11 mL) andwater (1 mL) which had been cooled at 0° C. and the resulting solutionwas stirred at 0° C. for 30 minutes, then warmed to room temperature.After 5 h, the reaction was found to be complete by HPLC. The reactionwas concentrated in vacuo and the residue was taken up in 25 mL MeOH(slurry) and concentrated. This process was repeated twice and theresidue was placed briefly on high vac. The material was taken up in 15mL MeOH and the suspension was filtered through a medium frit. Thefiltrate was concentrated in vacuo, the residue was taken up in 40 mL20% MeOH/EA and the solution was washed with 25 mL sat NaHCO₃. Theaqueous layer was back extracted once with 10 mL EA and the combinedorganics were dried over Na₂SO₄. The solution was filtered andconcentrated to yield a glass/foam. The product was isolated bypreparative TLC (two 20 cm×20 cm×1.0 mm prep TLC plates, 15% 7N NH₃ inCH₃OH/CH₂Cl₂) to yield the title compound (90 mg, 43%) as a foam: ¹H NMR(400 MHz, d₄-MeOH) ppm 8.08 (s, 1H), 7.26 (m, 3H), 7.21 (m, 2H), 6.63(d, J=3.52 Hz, 1H), 6.12 (d, J=4.56 Hz, 1H), 4.49 (t, J=4.87 Hz, 1H),4.21 (m, 1H), 4.17 (t, J=5.60 Hz, 1H), 3.22 (t, J=6.43 Hz, 2H), 2.96 (m,2H), 2.72 (t, J=6.95 Hz, 2H), 2.46 (s, 3H), 1.76 (m, 2H), 1.28 (s, 9H);MS (ESI+) for C₂₆H₃₇N₇O₄ m/z 512.2 (M+H)⁺; (ESI−) for C₂₆H₃₇N₇O₄ m/z510.2 (M−H)⁻; HPLC purity 97% (ret. time, 2.81 min).

Step 9:1-(4-(tert-butyl)phenyl)-3-(3-((((2R,3S,4R,5R)-5-(4-((2,4-dimethoxybenzyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)(methyl)amino)propyl)ureahydrochloride

A solution of1-(4-(tert-butyl)phenyl)-3-(3-((((2R,3S,4R,5R)-5-(4-((2,4-dimethoxybenzyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)(methyl)amino)propyl)urea(20 mg, 0.039 mmol) in methanol (0.7 mL) was added to a mixture of 0.1NHCl (0.39 mL, 0.039 mmol) and water (2.0 mL). The colorless solution wasconcentrated in vacuo to remove the methanol. The solution waslyophilized to yield the title compound (20 mg, 93%) as a white solid:¹H NMR (400 MHz, D₂O) ppm 8.02 (s, 1H), 7.33 (d, 1=3.73 Hz, 1H), 7.21(d, J=8.09 Hz, 2H), 6.90 (d, J=8.09 Hz, 2H), 6.69 (d, J=3.73 Hz, 1H),6.13 (d, J=4.56 Hz, 1H), 4.47 (m, 2H), 4.24 (t, J=5.39 Hz, 1H), 3.67 (m,1H), 3.54 (d, J=12.02 Hz, 1H), 3.33 (m, 1H), 3.22 (m, 1H), 3.11 (m, 1H),2.94 (s, 3H), 1.90 (m, 2H), 1.20 (s, 9H); MS (ESI+) for C₂₆H₃₇N₇O₄ m/z512.3 (M+H)⁺; (ESI−) for C₂₆H₃₇N₇O₄ m/z 510.2 (M−H)⁻; HPLC purity 97%(ret. time, 2.82 min). IC₅₀<10 nM.

Preparation of Compounds 16 and 17

The following steps are graphically depicted in FIG. 4.

Step 1:7-((3aR,4R,6R,6aR)-6-(azidomethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-N-(2,4-dimethoxybenzyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

A solution of((3aR,4R,6R,6aR)-6-(4-((2,4-dimethoxybenzyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methanol(2.83 g, 6.20 mmol) and triphenylphosphine (2.28 g, 8.68 mmol) in drytetrahydrofuran (32 mL) was cooled at 0° C. in an ice/water bath.Diisopropyl azodicarboxylate (1.71 mL, 8.68 mmol) was added dropwise,followed by a solution of diphenylphosphonic azide (1.87 mL, 8.68 mmol)in tetrahydrofuran (5.3 mL, 66 mmol). Upon addition of the DPPAsolution, a white milky precipitate formed. After about 30 minutes, thereaction mixture was allowed to warm to room temperature and stirovernight. After 24 h, HPLC indicated that all the starting material hadbeen consumed. The reaction mixture was concentrated to about ½ theoriginal volume and purified by flash chromatography (175 g silica gel,10-55% EA/hept) to yield the title compound (2.49 g, 83%) as a slightlyyellow stiff foam: MS (ESI+) for C₂₃H₂₇N₇O₅ m/z 482.2 (M+H)⁺; (ESI−) forC₂₃H₂₇N₇O₅ m/z 480.1 (M+H) m/z 526.1 (M+CO₂H)⁻; HPLC purity 97% (ret.time, 3.64 min).

Step 2:7-((3aR,4R,6R,6aR)-6-(aminomethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-N-(2,4-dimethoxybenzyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

A solution of((3aR,4R,6R,6aR)-6-(azidomethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-N-(2,4-dimethoxybenzyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine(2.49 g, 5.17 mmol) in tetrahydrofuran (50 mL, 600 mmol) was treateddropwise with a solution of 1.0 M of trimethylphosphine intetrahydrofuran (7.24 mL, 7.24 mmol) and the mixture was stirred at roomtemperature overnight. After 20 h all starting material was consumed byHPLC. The reaction mixture was treated with water (1.80 mL, 99.9 mmol)and stirred at rt for 2 h. The reaction mixture was concentrated, thecrude product was taken up in 90 mL CH₂Cl₂ and washed with four 30 mLportions of H₂O and 15 mL brine. The solution was dried over Na₂SO₄,filtered and concentrated to yield an oil that under the application ofa high vacuum became a foam. The crude material was purified by flashchromatography (120 g silica gel, 3-10% 7N NH₃ in CH₃OH/CH₂Cl₂) to yieldthe title compound (136 g, 75%) as a foam: MS (ESI+) for C₇₃H₂₉N₅O₅ m/z456.2 (M+H)⁺; (ESI−) for C₂₆H₃₅N₅O₅ m/z 454.1 (M−H)⁻; HPLC purity 92%(ret. time, 2.65 min).

Step 3:N-(2,4-dimethoxybenzyl)-7-((3aR,4R,6R,6aR)-6-((isopropylamino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

A solution of((3aR,4R,6R,6aR)-6-(aminomethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-N-(2,4-dimethoxybenzyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine(1.76 g, 3.86 mmol) in 1,2-dichloroethane (34 mL) was treated withacetone (0.31 mL, 42 mmol) and acetic acid (0.22 mL, 3.9 mmol) dropwisefollowed by sodium triacetoxyborohydride (0.98 g, 4.6 mmol) and themixture was stirred at room temperature till complete. After 1 h, HPLCindicated the starting material had been consumed and the reaction wascomplete. The reaction mixture was diluted with 60 mL CH₂Cl₂ and washedwith 50 mL sat NaHCO₃. The aqueous phase was washed with 30 mL CH₂Cl₂and the combined organic phase was washed with 40 mL brine and driedover Na₂SO₄. The solution was filtered and concentrated to yield thetitle compound (1.76 g, 92%) as a glass that was used directly in thenext step: MS (ESI+) for C₂₆H₃₅N₅O₅ m/z 498.3 (M+H)⁺; HPLC purity 90%(ret. time, 2.74 min).

Step 4:2-(3-((((3aR,4R,6R,6aR)-6-(4-((2,4-dimethoxybenzyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)(isopropyl)amino)propyl)isoindoline-1,3-dione

A mixture of γ-bromopropylphthalimide (2.37 g, 8.85 mmol),tetra-n-butylammonium iodide (0.234 g, 0.632 mmol).N,N-diisopropylethylamine (1.40 mL, 8.04 mmol) andN-(2,4-dimethoxybenzyl)-7-(3aR,4R,6R,6aR)-6-((isopropylamino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine(3.42 g, 6.32 mmol) was taken up in propanenitrile (25 mL) and washeated at 95° C. After 48 h at 95° C., HPLC indicated that the reactionwas nearly complete. The reaction mixture was cooled to roomtemperature, the mixture was diluted with 200 mL ethyl acetate andwashed with two 100 mL portions of H₂O and 100 mL brine. The organicphase was dried over Na₂SO₄, filtered and concentrated to yield a glass.The crude material was purified by flash chromatography (250 g silicagel, 2-4% 7N NH₃ in CH₃OH/CH₂Cl₂) to yield the title compound (3.12 g,72%) as a foam: MS (ESI+) for C₃₇H₄₄N₆O₇ m/z 685.2 (M+H)⁺, (ESI−) forC₃₇H₄₄N₆O₇ m/z 729 (M+HCO₂)⁻; HPLC purity 99% (ret. time, 3.17 min).

Step 5:N¹-(((3aR,4R,6R,6aR)-6-(4-((2,4-dimethoxybenzyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-N¹-isopropylpropane-1,3-diamine

2-(3-((((3aR,4R,6R,6aR)-6-(4-((2,4-dimethoxybenzyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)(isopropyl)amino)-propyl)isoindoline-1,3-dione(1.37 g, 2.00 mmol) was dissolved in 2M methylamine in methanol (30 mL,60 mmol). The solution was stirred at room temperature for 5 minutesthen heated at 55-60° C. After 1 h, the SM was consumed by HPLC. Thereaction mixture was cooled to room temperature and concentrated invacuo. The resultant tan oil was taken up in 20 mL MeOH andconcentrated. The procedure was repeated to an oil. The material wasplaced on high vacuum to yield a solid which contained the titlecompound along with N-methylphthalimide and was used as is in the nextstep: MS (ESI+) for C₂₉H₄₂N₆O₅ m/z 555.4 (M+H)⁺; HPLC ret. time 2.57min.

Step 6:1-(4-(tert-butyl)phenyl)-3-(3-((((3aR,4R,6R,6aR)-6-(4-((2,4-dimethoxybenzyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)(isopropyl)amino)propyl)urea

A suspension ofN¹-(((3aR,4R,6R,6aR)-6-(4-((2,4-dimethoxybenzyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-N¹-isopropylpropane-1,3-diamine(1.11 g, 2.00 mmol, crude from step 6) in methylene chloride (40 mL) wastreated dropwise with a solution of 1-tert-butyl-4-isocyanatobenzene(0.36 mL, 2.0 mmol) in methylene chloride (3.5 mL) and allowed to stirat room temperature. After 1 h, reaction was complete by HPLC. Thereaction mixture was concentrated to yield a glass. The crude materialwas purified by flash chromatography (100 g silica gel, 2-4% 7N NH₃ inCH₃OH/CH₂Cl₂ to yield the title compound (1.07 g, 73%) as a foam: MS(ESI+) for C₁₀H₅₅N₇O₆ m/z 730.4 (M+H)⁺; (ESI−) for C₄₀H₅₅N₇O₆ m/z 728.5(M−H)⁻; HPLC purity, 89% (ret. time, 3.78 min).

Step 7:1-(3-((((2R,3S,4R,5R)-5-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)(isopropyl)amino)propyl)-3-(4-(tert-butyl)phenyl)urea

1-(4-(tert-butyl)phenyl)-3-(3-((((3aR,4R,6R,6aR)-6-(4-((2,4-dimethoxybenzyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)(isopropyl)amino)propyl)urea(1.07 g, 1.39 mmol) was dissolved in a mixture of trifluoroacetic acid(25 mL) and water (2.5 mL) which had been cooled at 0° C. and theresulting solution was stirred at 0° C. for 30 minutes, then warmed toroom temperature. After 4 h, the reaction was found to be complete byHPLC. The reaction mixture was concentrated in vacuo and the residue wastaken up in 25 mL MeOH (white slurry) and concentrated. This process wasrepeated three times and the resultant residue was placed under highvacuum. The material was taken up in 100 mL 10% MeOH/CH₂Cl₂ and washedwith two 75 mL portions of sat NaHCO3 and 50 mL 1% aq Na₂CO₃. Theorganic phase was dried over Na₂SO₄, filtered and concentrated to yielda glass/solid. The crude material was purified by flash chromatography(100 g silica gel, 5-10% 7N NH₃ in CH₃OH/CH₂Cl₂) to yield the titlecompound (0.35 g, 46%) as a colorless glass: MS (ESI+) for C₂₈H₄₁N₇O₄m/z 540.3 (M+H)⁺; (ESI−) for C₂₈H₄₁N₇O₄ m/z 538.3 (M−H)⁻, m/z 584.4(M+HCO₂)⁻; HPLC purity 98% (ret. time 2.86 min); ¹H NMR (400 MHz,d₄-MeOH) ppm 8.05 (s, 1H), 7.27 (d, J=3.73 Hz, 1H), 7.24 (m, 2H), 7.18(m, 2H), 6.63 (d, J=3.73 Hz, 1H), 6.15 (d, J=4.77 Hz, 1H), 4.46 (t,J=5.08 Hz, 1H), 4.18 (t, J=5.39 Hz, 1H), 4.11 (m, 1H), 3.22 (m, 2H),3.07 (m, 1H), 2.85 (m, 1H), 2.72 (m, 1H), 2.60 (t, J=6.43 Hz, 2H), 1.68(m, 2H), 1.28 (s, 9H), 1.05 (d, J=6.63 Hz, 3H), 1.01 (d, J=6.43 Hz, 3H).

Step 8:1-(3-((((2R,3S,4R,5R)-5-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)(isopropyl)amino)propyl)-3-(4-(tert-butyl)phenyl)ureahydrochloride

A solution of1-(3-((((2R,3S,4R,5R)-5-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)(isopropyl)amino)propyl)-3-(4-(tert-butyl)phenyl)urea(1.64 g, 3.04 mmol) in 50 mL 50% aq methanol was treated with 1.0N ofhydrogen chloride in water (3.87 mL, 3.04 mmol). The solution wasconcentrated to remove most of the methanol and lyophilized overnight.The cloudy mixture was filtered through a fine frit and the filtrate wasconcentrated in vacuo to remove the MeOH. The resultant solution waslyophilized overnight to yield the title compound (1.70 g, 97%) as asolid: MS (ESI+) for C₂₈H₄₁N₇O₄ m/z 540.4 (M+H)⁺; MS (ESI+) forC₂₈H₄₁N₇O₄ m/z 538.4 (M+H)⁺, m/z 574.4 (M+Cl)⁻; HPLC purity 97% (ret.time, 2.88 min); ¹H NMR (400 MHz, d₄-MeOH) ppm 8.12 (s, 1H), 7.29 (m,2H), 7.23 (m, 3H), 6.68 (m, 1H), 6.09 (br. s., 1H), 4.57 (m, 1H), 4.35(m, 2H), 3.79 (br. s., 1H), 3.55 (m, 2H), 3.26 (br. s., 4H), 1.94 (m,2H), 1.35 (m, 6H), 1.29 (s, 9H). IC₅₀<10 nM.

Preparation of Compound 18(2R,3R,4S,5R)-2-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-(((4-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)butyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol

General

HPLC conditions: Agilent 1100 HPLC. Zorbax Eclipse XDB-C18 50×4.6 mmcolumn. Solvent A—Water (0.1% TFA); Solvent B—Acetonitrile (0.07% TFA).Flow rate—1.50 mL/min. Gradient—5 min 95% A to 90% B, 1 min hold, thenrecycle (to 95% A over 1 min). UV detection @ 214 and 254 nm.

Step 1: Benzyl 5-bromopentanoate

A solution of 5-bromopentanoic acid (1.00 g, 5.52 mmol) and benzylalcohol (0.286 mL, 2.76 mmol) in methylene chloride (14 mL) was treatedsequentially with N,N′-diisopropylcarbodiimide (0.523 g, 4.14 mmol) and4-dimethylaminopyridine (43.9 mg, 0.359 mmol). The solution was allowedto stir at room temperature. A precipitate formed within 1 minute of theaddition of the 4-dimethylaminopyridine. After ˜65 h, the reactionmixture was filtered, the solid was washed with CH₂Cl₂ and the filtratewas washed with 20 mL portions of 1N HCl, sat NaHCO₃ and brine and driedover Na₂SO₄. The solution was filtered and concentrated to yield anearly colorless liquid along with a solid (DIC urea). The material wastaken up in 1/1 MTBE/heptane to yield a solution with a whiteprecipitate. The mixture was filtered and concentrated and the crudematerial was purified by flash chromatography (75 g silica gel; 5-10%MTBE/hept) to yield the title compound (0.69 g, 92%) as a colorlessliquid. HPLC purity>95° A) (ret. time, 4.73 min).

Step 2: Benzyl5-((((3aR,4R,6R,6aR)-6-(4-((2,4-dimethoxybenzyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)(isopropyl)amino)pentanoate

A mixture of benzyl 5-bromopentanoate (116 mg, 0.427 mmol),tetra-n-butylammonium iodide (11.3 mg, 0.0305 mmol),N,N-diisopropylethylamine (69.08 uL, 0.3966 mmol) andN-(2,4-dimethoxybenzyl)-7-(3aR,4R,6R,6aR)-6-((isopropylamino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine(165 mg, 0.305 mmol) was taken up in propanenitrile (1.0 mL) to give alight tan solution that was heated at 95° C. After 68 h at 95° C., HPLCindicated the starting material had been consumed. The reaction mixturewas cooled to room temperature, diluted with 30 mL ethyl acetate andwashed with two 25 mL portions of H₂O and 25 mL brine. The organic phasewas dried over Na₂SO₄, filtered and concentrated to yield a tan viscousglass. The crude material was purified by flash chromatography (40 gsilica gel; 1.5% 7N NH₃ in CH₃OH/CHCl₃) to yield the title compound (151mg, 72%) as a colorless glass: MS (ESI+) for C₃₈H₄₉N₅O₇ m/z 688.3(M+H)⁺; MS (ESI−) for C₃₈H₄₉N₅O₇ m/z 686.7 (M−H)⁻; HPLC purity 95% (ret.time, 3.48 min).

Step 3:5-(((((3aR,4R,6R,6aR)-6-(4-((2,4-dimethoxybenzyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)(isopropyl)amino)pentanoicacid

A solution of benzyl5-((((3aR,4R,6R,6aR)-6-(4-((2,4-dimethoxybenzyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)(isopropyl)amino)pentanoate(151 mg, 0.220 mmol) in ethanol (4.1 mL) was treated with 10% palladiumon carbon (51 mg, 0.048 mmol) and 1,4-cyclohexadiene (0.23 mL, 2.4mmol). The mixture was heated at 85° C. until the starting material wasconsumed as indicated by HPLC. After about 1 h, the starting materialwas consumed as indicated by HPLC and the reaction mixture was cooled toroom temperature. The mixture was filtered through a pad of celite andthe pad was washed with 40 mL EtOH. The solution was concentrated toyield the title compound (136 mg, 104%) as a nearly colorless glass,which was taken up in toluene, concentrated and placed under high vac.The material was determined to be of sufficient purity to be used in thenext step without purification: MS (ESI+) for C₃₁H₄₃N₅O₇ m/z 598.7(M+H)⁺; HPLC purity>95% (ret. time, 2.84 min).

Step 4:N-(2-amino-4-(tert-butyl)phenyl)-5-((((3aR,4R,6R,6aR)-6-(4-((2,4-dimethoxybenzyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)(isopropyl)amino)pentanamide

A solution of5-((((3aR,4R,6R,6aR)-6-(4-((2,4-dimethoxybenzyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)(isopropyl)amino)pentanoicacid (131 mg, 0.219 mmol) and 4-tert-butylbenzene-1,2-diamine (40 mg,0.24 mmol) in N,N-dimethylformamide (2.2 mL) was treated withN,N-diisopropylethylamine (84 uL, 0.48 mmol) dropwise followed by PyBopreagent (120 mg, 0.24 mmol). The solution was allowed to stir at roomtemperature for 19 h, whereupon HPLC indicated the reaction wascomplete. The reaction mixture was concentrated under high vacuum. Theresidue was taken up in 40 mL CH₂Cl₂ and washed with 20 mL portions ofH₂O, 5% citric acid, and brine. The organic phase was dried over Na₂SO₄,filtered and concentrated to yield a tan viscous glass. The crudematerial was purified by flash chromatography (40 g silica gel; 2% 7NNH₃ in CH₃OH/CH₂Cl₂ to yield the title compound (140 mg, 86%) was aslightly tan glass: MS (ESI+) for C₄₁H₅₇N₇O₆ m/z 744.9 (M+H)⁺; HPLCpurity (combined for the two regioisomers) 91% (ret. times, 3.25 and3.28 min).

Step 5:7-((3aR,4R,6R,6aR)-6-(((4-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)butyl)(isopropyl)amino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-N-(2,4-dimethoxybenzyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

N-(2-amino-4-(tert-butyl)phenyl)-5-((((3aR,4R,6R,6aR)-6-(4-((2,4-dimethoxybenzyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)(isopropyl)amino)pentanamide(140 mg, mmol) was taken up in acetic acid (3.5 mL) and the solution washeated at 65° C. for 2 h, whereupon HPLC indicated the starting materialhad been consumed. The reaction mixture was cooled to room temperatureand the solvent was removed under high vac. The residue was taken up in30 mL CH₂Cl₂ and washed with 20 mL portions of sat NaHCO₃ and 2% Na₂CO₃solution. The organic phase was dried over Na₂SO₄, filtered andconcentrated to yield a light tan glass/stiff foam. The crude materialwas purified by flash chromatography (25 g silica gel; 3% 7N NH₃ inCH₃OH/CH₂Cl₂) to yield the title compound (120 mg, 88%) as a slightlytan glass: MS (ESI+) for C₄₁H₅₅N₇O₅ m/z 726.5 (M+H)⁺; MS (ESI−) forC₄₁H₅₅N₇O₅ m/z 724.6 (M−H)⁻; HPLC purity 90% (ret. time, 3.14 min).

Step 6:(2R,3R,4S,5R)-2-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-((4-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)butyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol

7-((3aR,4R,6R,6aR)-6-(((4-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)butyl)(isopropyl)amino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-N-(2,4-dimethoxybenzyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine(120 mg, 0.16 mmol) was dissolved in a mixture of trifluoroacetic acid(5.0 mL) and water (0.5 mL) which had been precooled at 0° C. in an icebath. The solution was stirred at 0° C. for 30 minutes, and then warmedto room temperature. After 4.5 h at room temperature, the reaction wasfound to be complete by HPLC and the now pink reaction mixture wasconcentrated. The residue was taken up in 10 mL MeOH and concentrated.This procedure was repeated twice and the residue placed on high vac for1 h. The material was taken up in 6 mL MeOH and was treated with 100 mgK₂CO₃ and five drops of water. The mixture was stirred for 30 minutesduring which time the mixture was found to be basic. The mixture wasfiltered through a fine frit, the solids were washed with 10 mL MeOH andthe filtrate was concentrated to yield a nearly colorless solid. Thecrude material was purified by flash chromatography (20 g silica gel; 8%7N NH₃ in CH₃OH/CH₂Cl₂) to yield the title compound (60 mg, 68%) as acolorless glass: MS (ESI+) for C₂₉H₄₁N₇O₃ m/z 536.5 (M+H)⁺; MS (ESI−)for C₂₇H₄₁N₇O₃ m/z 534.5 (M−H)⁻; HPLC purity>95% (ret. time, 2.53 min);¹H NMR (400 MHz, d4-MeOH) δ 8.07 (s, 1H), 7.48 (br. s., 1H), 7.38 (d,J=8.50 Hz, 1H), 7.27 (dd, J=8.50, 1.87 Hz, 1H), 7.22 (d, J=3.73 Hz, 1H),6.61 (d, J=3.73 Hz, 1H), 6.11 (d, J=4.77 Hz, 1H), 4.43 (t, J=5.08 Hz,1H), 4.14 (t, J=5.49 Hz, 1H), 4.04 (m, 1H), 3.00 (m, 1H), 2.83 (m, 3H),2.67 (dd, J=14.10, 6.84 Hz, 1H), 2.53 (m, 2H), 1.80 (m, 2H), 1.52 (m,2H), 1.36 (s, 9H), 1.01 (d, J=6.63 Hz, 3H), 0.97 (d, J=6.63 Hz, 3H).

Compounds 20 and 21 were synthesized by analogous procedures. See FIGS.7 and 8, respectively.

Inhibition of DOT1L

Compounds 9 and 17 are potent inhibitors of DOT1L in biochemical assays(see Table 1, below). To evaluate the ability of this compound toinhibit DOT1L in cells, its effect on cellular histone H3 lysine 79(H3K79) methylation was examined. DOT1L is the only known histonemethyltransferase capable of methylating H3K79, and so inhibition ofcellular DOT1L should lead to a reduction of cellular H3K79 methylation.

TABLE 1 H3K79 IC₅₀ Proliferation IC₅₀ Biochemical IC₅₀ MOLM-13 cellsMOLM-13 cells Compound # (nM) (nM) (μM) 9 6 77 17.3 17 <1 59 3.2 18 <1020 <1,000 21 <1,000

The data in Table 1 indicates that 9 and 17 can enter cells and inhibitDOT1L in a cellular context. The compound 9 and 17 also inhibitedproliferation in the cells.

In summary, inhibition of DOT1L activity with 9 and 17 leads todepletion of H3K79 methylation and a dramatic decrease in growth andviability of MLL-rearranged leukemia cell lines.

Selective Killing of Mixed Lineage Leukemia Cells by Compound 16, APotent Small-Molecule DOT1L Inhibitor

Based on the chemical structures of the SAM substrate andS-adenosylhomocysteine (SAH) product, the reaction mechanism of DOT1Lcatalysis and the published crystal structure of the DOT1L active site,medicinal chemistry design tenets were established to facilitatemechanism-guided inhibitor discovery; chemical analogues thus designedwere synthesized and tested as inhibitors of DOT1L enzymatic activity.From these efforts, compound 16 was identified. This compounddemonstrates potent, concentration-dependent inhibition of DOT1L enzymeactivity with an 1050 of 400±100 pM. The chemical structure of thiscompound retains the nucleoside core of the SAM substrate, and SAHproduct. As such it was designed to bind to the enzyme within the SAMbinding pocket. Steady state kinetic analysis confirms that the compoundbinds to the enzyme competitively with SAM. For example, adistinguishing feature of competitive inhibition is a linear increase inthe apparent IC50 of the compound as a function of substrateconcentration; compound 16 displays this pattern when assayed as afunction of SAM concentration relative to the KM of SAM. SAM is a commonmethyl group donator that is used by all histone methyltransferases(HMTs). Despite the universality of SAM utilization by HMTs, compound 16displays remarkable selectivity for inhibition of DOT1L over other HMTs,as summarized in FIG. 5. Thus, the compound displays a minimumselectivity of >1000-fold for DOT1L relative to all HMTs that have beentested.

Compound 16 Selectively Inhibits Cellular H3K79 Methylation

Having established that compound 16 is a potent and highly selectiveDOT1L inhibitor in biochemical assays, we next tested the ability ofcompound 16 to inhibit DOT1L in cells by immunoblot analysis ofextracted histones using an antibody specific for dimethylated H3K79(H3K79me2). Treatment of human cell lines derived from MLL-rearrangedacute myeloid leukemia (AML) (MOLM-13, MLL-AF9), MLL-rearrangedbiphenotypic leukemia (MV4-11, MLL-AF4), or non-MLL-rearranged T-cellacute leukemia (Jurkat) with compound 16 led to a concentrationdependent reduction in global H3K79me2 levels. To understand thekinetics of compound 16-mediated cellular H3K79me2 depletion, weperformed a time course analysis in MV4-11 cells incubated with 3 μMcompound 16, a concentration sufficient for maximal cellular DOT1Linhibition. A modest reduction in H3K79me2 levels was apparent withinone day of treatment, but full depletion took four to five days. Thereis no known histone demethylase enzyme specific for H3K79, so thedecline in methylation at this residue following DOT1L inhibition ispresumably due to incorporation into chromatin of unmethylated H3through histone turnover and replacement.

To assess the specificity of compound 16 inhibitory activity in cells,we immunoblotted histones extracted from compound 16-treated MV4-11cells with a panel of methyl-lysine and methyl-arginine residue specificantibodies. The only methyl marks affected by compound 16 treatment wereH3K79me1 and H3K79me2, consistent with compound 16 being a highlyspecific DOT1L inhibitor in a cellular context.

Compound 16 Blocks MLL Fusion Target Gene Expression

We next tested whether compound 16 was able to inhibit expression of keyMLL fusion target genes. HOXA9 and MEIS1 over expression is a hallmarkof MLL, rearranged leukemias (Armstrong et al., 2002; Ferrando et al.,2003; Ross et al., 2004; Ross et al., 2003; Rozovskaia et al., 2001;Yeoh et al., 2002). Furthermore, both genes are bound by MLL fusionproteins, hypermethylated at H3K79 and down-regulated by DOT1L RNAiknockdown in MLL-rearranged cell lines, including MV4-11 (Guenther etal., 2008; Krivtsov et al., 2008; Lin et al., 2010; Milne et al., 2005;Monroe et al., 2010; Mueller et al., 2009; Okada et al., 2005; Thiel etal.; Yokoyama et al., 2010). We used quantitative real-time PCR (RT-PCR)to examine the effect of compound 16 on HOXA9 and MEIS1 transcriptlevels in MOLM-13 and MV4-11 cells. Treatment with compound 16 led to aconcentration-dependent decrease of both transcripts in each cell linewith IC50s of approximately 700 nM. We evaluated the kinetics of thisdecrease by measuring HOXA9 and MEIS1 mRNA levels over time in cellstreated with 3 μM compound 16. Levels of both transcripts weresignificantly decreased within 48 hours of compound addition, and weremaximally reduced after six to eight days of compound 16 treatment(fitting of these data yielded estimated half-lives of 2.3 and 3.3 daysfor HOXA9 and MEIS1 inhibition, respectively). This decrease was not dueto a general inhibitory effect on gene expression since transcriptlevels of the housekeeping gene TBP were unaffected.

Compound 16 Selectively Inhibits Proliferation of MLL-Rearranged Cells

Having established that compound 16 can inhibit H3K79 methylation andblock MLL fusion target gene expression, we investigated whether thistranslated into anti-proliferative activity in MLL-rearranged leukemiccells. We performed proliferation assays over several days with MV4-11and MOLM-13 cells in the presence or absence of 3 μM compound 16. Jurkatcells were included as a non-MLL rearranged cell line control. Theeffect of extended compound 16 treatment was remarkably specific for theMLL-rearranged cell lines. The number of viable MV4-11 and MOLM-13 cellswas dramatically reduced by compound 16, whereas the growth of Jurkatcells was unaffected. The lack of effect on Jurkat cells was not due todifferences in the ability of compound 16 to inhibit DOT1L in thesecells as measured by immunoblot for cellular H3K79me2 levels. Thisanalysis also revealed a significant delay before the antiproliferativeeffects of compound 16 became apparent; both MLL-rearranged cell linescontinued to proliferate at a normal rate for several days afterexposure to the inhibitor. This may reflect the time required to reversefully the aberrant expression of MLL fusion target genes following DOT1Linhibition, a process that presumably involves depletion of methylatedH3K79, followed by decreased mRNA expression and reduced levels of geneproducts critical for leukemogenic growth. To expand our analysis of thedifferential sensitivity of MLL-rearranged cell lines to compound 16, wedetermined IC50 values for inhibition of proliferation in a panel of sixMLL-rearranged and six non-rearranged human leukemia cell lines. The MLLrearranged panel (FIG. 6) included human cell lines derived from ALL,AML and biphenotypic leukemias harboring MLL-AF4, MLL-AF9 or MLL-ENLfusions. As shown in FIG. 6, IC50 values for MLL-rearranged cell lineswere in the nanomolar to low micromolar range, whereas IC50s for non-MLLrearranged cell lines were always above 10 μM or undetermined due tolack of inhibition at the highest concentration tested (reported asIC50>50 μM in FIG. 6). We next determined whether these results wouldextend to primary murine hematopoietic progenitors transformed byretroviral expression of an MLL-AF9 fusion protein.

These results demonstrate that DOT1L methyltransferase activity isrequired for proliferation of MLL-rearranged cells and MLL fusionmediated transformation, but is not essential for proliferation andviability of non-MLL-rearranged cells in culture.

Compound 16 Causes Differentiation and Apoptosis in MLL-Rearranged Cells

To explore the mechanism of cell killing in more detail, we determinedeffects of compound 16 on the cell cycle and apoptosis in MV4-11 andMOLM-13 cells by flow cytometry for DNA content and Annexin V staining.In MV4-11 cells, a modest increase in G0/G1 phase, and a decrease inS-phase cells were apparent after four days of incubation with 3 μMcompound 16. This was followed by an increase in sub-G1 andAnnexin-positive cells over the next six days, consistent with apoptoticcell death. Similar results were obtained in MOLM-13 cells, although thepercentage of Annexin-positive cells was significantly lower. We nextanalyzed whether compound 16 induced differentiation prior to celldeath. MOLM-13 cells were treated with 3 μM compound 16 and monitoredfor cell surface expression of the myeloid differentiation marker CD14by flow cytometry. Expression of CD14 was induced following 12 days ofcompound 16 treatment. Gene set enrichment analysis (GSEA) (Subramanianet al., 2005) of genes upregulated by compound 16 treatment of MOLM-13cells (see below) also demonstrated significant enrichment forhematopoietic cell lineage markers, including CD14, (NormalizedEnrichment Score (NES)=1.78, False Discovery Rate (FDR)=0.054). Thisprovides further evidence that small-molecule inhibition of DOT1Lpromotes some degree of differentiation prior to cell killing.

Compound 16 Reverses the MLL-rearranged Gene Signature

To determine effects of compound 16 treatment on gene expression in MLLrearranged leukemia cell lines, RNA was isolated from MV4-11 cells andMOLM-13 cells treated with 3 μM compound 16 for up to six days,amplified and hybridized to Affymetrix microarrays. Statisticallysignificant changes in gene expression (probes with statisticallysignificant changes (q<0.15) and up or down-regulated at least 2-fold)were not observed until four days after inhibitor treatment, consistentwith the relatively delayed effects of compound 16 on H3K79 methylationand proliferation. Among the genes down-regulated in MV4-11 and MOLM-13cells following 6 days of compound 16 treatment are several that havebeen previously implicated in MLL fusion mediated leukemogenesisincluding multiple HOXA genes, MEIS1 and MEF2C. GSEA of genesdown-regulated following 6 day compound 16 treatment of MOLM-13 cellsdemonstrated strong enrichment (NES=−1.74, FDR=0.014) for genes overexpressed in MLL-rearranged human acute leukemias as compared toMLL-germline acute leukemias (Ross et al., 2004). This indicates thatsmall-molecule inhibition of DOT1L is able to reverse the MLL-rearrangedgene expression signature in MLL-rearranged cell lines. We next usedGSEA to compare genes downregulated following 6 day compound 16treatment of MOLM-13 (MLL-AF9) or MV4-11 (MLL-AF4) cells with genesidentified as direct targets of MLL-AF9, or MLLAF4 (Guenther et al.,2008) through genome-wide chromatin immunoprecipitation coupled withlarge scale sequencing (ChIP-seq). Genes down-regulated by compound 16in MOLM-13 cells were significantly enriched for direct MLL-AF9 targets(NES=−1.86, FDR: 0.007), whereas genes down-regulated by compound 16 inMV4-11 cells were enriched for direct MLL-AF4 targets (NES=−1.51, FDR:0.081, FIG. 5D). Both results indicate that small-molecule inhibition ofDOT1L decreases the expression of direct MLL fusion targets. Finally, wecompared gene expression changes caused by compound 16 treatment ofMOLM-13 (MLL-AF9) cells with those caused by genetic knockout of Dot1Lin a mouse model of MLL-AF9 leukemia. We found significant overlapbetween these gene expression changes (NES=−1.58, FDR: 0.024, FIG. 5E)indicating that compound 16 treatment and genetic ablation of Dot1Lcause cell killing of MLL-rearranged cells through similar pathways.

INCORPORATION BY REFERENCE

All of the U.S. patents and U.S. published patent applications citedherein are hereby incorporated by reference.

EQUIVALENTS

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto; the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

We claim:
 1. A compound selected from

and pharmaceutically acceptable salts, hydrates, enantiomers andstereoisomers thereof.
 2. A compound


3. A pharmaceutically acceptable salt of


4. The pharmaceutically acceptable salt of claim 3, being


5. A pharmaceutical composition comprising a compound of claim 1 and apharmaceutically acceptable diluent or carrier.
 6. A pharmaceuticalcomposition comprising a compound of claim 2 and a pharmaceuticallyacceptable diluent or carrier.
 7. A pharmaceutical compositioncomprising a pharmaceutically acceptable salt of claim 3 and apharmaceutically acceptable diluent or carrier.
 8. A pharmaceuticalcomposition comprising a pharmaceutically acceptable salt of claim 4 anda pharmaceutically acceptable diluent or carrier.
 9. A kit comprising acompound of claim 1 and instructions for use thereof.
 10. A kitcomprising a compound of claim 2 and instructions for use thereof.
 11. Akit comprising a pharmaceutically acceptable salt of claim 3 andinstructions for use thereof.
 12. A kit comprising a pharmaceuticallyacceptable salt of claim 4 and instructions for use thereof.